Methods and compositions for cns delivery of arylsulfatase a

ABSTRACT

The present invention provides, among other things, compositions and methods for CNS delivery of lysosomal enzymes for effective treatment of lysosomal storage diseases. In some embodiments, the present invention includes a stable formulation for direct CNS intrathecal administration comprising an arylsulfatase A (ASA) protein, salt, and a polysorbate surfactant for the treatment of Metachromatic Leukodystrophy Disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 61/358,857 filed Jun. 25, 2010; 61/360,786, filed Jul. 1,2010; 61/387,862, filed Sep. 29, 2010; 61/435,710, filed Jan. 24, 2011;61/442,115, filed Feb. 11, 2011; 61/476,210, filed Apr. 15, 2011 and61/495,268 filed on Jun. 9, 2011; the entirety of each of which ishereby incorporated by reference.

This application relates to US applications entitled “CNS Delivery ofTherapeutic Agents,” filed on even date; “Methods and Compositions forCNS Delivery of Heparan N-Sulfatase,” filed on even date; “Methods andCompositions for CNS Delivery of Iduronate-2-Sulfatase,” filed on evendate; “Methods and Compositions for CNS Delivery ofβ-Galactocerebrosidase,” filed on even date; “Treatment of SanfilippoSyndrome Type B,” filed on even date; the entirety of each of which ishereby incorporated by reference.

BACKGROUND

Enzyme replacement therapy (ERT) involves the systemic administration ofnatural or recombinantly-derived proteins and/or enzymes to a subject.Approved therapies are typically administered to subjects intravenouslyand are generally effective in treating the somatic symptoms of theunderlying enzyme deficiency. As a result of the limited distribution ofthe intravenously administered protein and/or enzyme into the cells andtissues of the central nervous system (CNS), the treatment of diseaseshaving a CNS etiology has been especially challenging because theintravenously administered proteins and/or enzymes do not adequatelycross the blood-brain barrier (BBB).

The blood-brain barrier (BBB) is a structural system comprised ofendothelial cells that functions to protect the central nervous system(CNS) from deleterious substances in the blood stream, such as bacteria,macromolecules (e.g., proteins) and other hydrophilic molecules, bylimiting the diffusion of such substances across the BBB and into theunderlying cerebrospinal fluid (CSF) and CNS.

There are several ways of circumventing the BBB to enhance braindelivery of a therapeutic agent including direct intra-cranialinjection, transient permeabilization of the BBB, and modification ofthe active agent to alter tissue distribution. Direct injection of atherapeutic agent into brain tissue bypasses the vasculature completely,but suffers primarily from the risk of complications (infection, tissuedamage, immune responsive) incurred by intra-cranial injections and poordiffusion of the active agent from the site of administration. To date,direct administration of proteins into the brain substance has notachieved significant therapeutic effect due to diffusion barriers andthe limited volume of therapeutic that can be administered.Convection-assisted diffusion has been studied via catheters placed inthe brain parenchyma using slow, long-term infusions (Bobo, et al.,Proc. Natl. Acad. Sci. U.S.A 91, 2076-2080 (1994); Nguyen, et al. J.Neurosurg. 98, 584-590 (2003)), but no approved therapies currently usethis approach for long-term therapy. In addition, the placement ofintracerebral catheters is very invasive and less desirable as aclinical alternative.

Intrathecal (IT) injection, or the administration of proteins to thecerebrospinal fluid (CSF), has also been attempted but has not yetyielded therapeutic success. A major challenge in this treatment hasbeen the tendency of the active agent to bind the ependymal lining ofthe ventricle very tightly which prevented subsequent diffusion.Currently, there are no approved products for the treatment of braingenetic disease by administration directly to the CSF.

In fact, many believed that the barrier to diffusion at the brain'ssurface, as well as the lack of effective and convenient deliverymethods, were too great an obstacle to achieve adequate therapeuticeffect in the brain for any disease.

Many lysosomal storage disorders affect the nervous system and thusdemonstrate unique challenges in treating these diseases withtraditional therapies. There is often a large build-up ofglycosaminoglycans (GAGs) in neurons and meninges of affectedindividuals, leading to various forms of CNS symptoms. To date, no CNSsymptoms resulting from a lysosomal disorder has successfully beentreated by any means available.

Thus, there remains a great need to effectively deliver therapeuticagents to the brain. More particularly, there is a great need for moreeffective delivery of active agents to the central nervous system forthe treatment of lysosomal storage disorders.

SUMMARY OF THE INVENTION

The present invention provides an effective and less invasive approachfor direct delivery of therapeutic agents to the central nervous system(CNS). The present invention is, in part, based on unexpected discoverythat a replacement enzyme (e.g., arylsulfatase A (ASA)) for a lysosomalstorage disease (e.g., MLD) can be directly introduced into thecerebrospinal fluid (CSF) of a subject in need of treatment at a highconcentration (e.g., greater than about 3 mg/ml, 4 mg/ml, 5 mg/ml, 10mg/ml or more) such that the enzyme effectively and extensively diffusesacross various surfaces and penetrates various regions across the brain,including deep brain regions. More surprisingly, the present inventorshave demonstrated that such high protein concentration delivery can bedone using simple saline or buffer-based formulations and withoutinducing substantial adverse effects, such as severe immune response, inthe subject. Therefore, the present invention provides a highlyefficient, clinically desirable and patient-friendly approach for directCNS delivery for the treatment various diseases and disorders that haveCNS components, in particular, lysosomal storage diseases. The presentinvention represents a significant advancement in the field of CNStargeting and enzyme replacement therapy.

As described in detail below, the present inventors have successfullydeveloped stable formulations for effective intrathecal (IT)administration of an arylsulfatase A (ASA) protein. It is contemplated,however, that various stable formulations described herein are generallysuitable for CNS delivery of therapeutic agents, including various otherlysosomal enzymes. Indeed, stable formulations according to the presentinvention can be used for CNS delivery via various techniques and routesincluding, but not limited to, intraparenchymal, intracerebral,intravetricular cerebral (ICV), intrathecal (e.g., IT-Lumbar,IT-cisterna magna) administrations and any other techniques and routesfor injection directly or indirectly to the CNS and/or CSF.

It is also contemplated that various stable formulations describedherein are generally suitable for CNS delivery of other therapeuticagents, such as therapeutic proteins including various replacementenzymes for lysosomal storage diseases. In some embodiments, areplacement enzyme can be a synthetic, recombinant, gene-activated ornatural enzyme.

In various embodiments, the present invention includes a stableformulation for direct CNS intrathecal administration comprising anarylsulfatase A (ASA) protein, salt, and a polysorbate surfactant. Insome embodiments, the ASA protein is present at a concentration rangingfrom approximately 1-300 mg/ml (e.g., 1-250 mg/ml, 1-200 mg/ml, 1-150mg/ml, 1-100 mg/ml, 1-50 mg/ml). In some embodiments, the ASA protein ispresent at or up to a concentration selected from 2 mg/ml, 3 mg/ml, 4mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml.

In various embodiments, the present invention includes a stableformulation of any of the embodiments described herein, wherein the ASAprotein comprises an amino acid sequence of SEQ ID NO:1. In someembodiments, the ASA protein comprises an amino acid sequence at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:1.In some embodiments, the stable formulation of any of the embodimentsdescribed herein includes a salt. In some embodiments, the salt is NaCl.In some embodiments, the NaCl is present as a concentration ranging fromapproximately 0-300 mM (e.g., 0-250 mM, 0-200 mM, 0-150 mM, 0-100 mM,0-75 mM, 0-50 mM, or 0-30 mM). In some embodiments, the NaCl is presentat a concentration ranging from approximately 137-154 mM. In someembodiments, the NaCl is present at a concentration of approximately 154mM.

In various embodiments, the present invention includes a stableformulation of any of the embodiments described herein, wherein thepolysorbate surfactant is selected from the group consisting ofpolysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 andcombination thereof. In some embodiments, the polysorbate surfactant ispolysorbate 20. In some embodiments, the polysorbate 20 is present at aconcentration ranging approximately 0-0.02%. In some embodiments, thepolysorbate 20 is present at a concentration of approximately 0.005%.

In various embodiments, the present invention includes a stableformulation of any of the embodiments described herein, wherein theformulation further comprises a buffering agent. In some embodiments,the buffering agent is selected from the group consisting of phosphate,acetate, histidine, succinate, Tris, and combinations thereof. In someembodiments, the buffering agent is phosphate. In some embodiments, thephosphate is present at a concentration no greater than 50 mM (e.g., nogreater than 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or5 mM). In some embodiments, the phosphate is present at a concentrationno greater than 20 mM. In various aspects the invention includes astable formulation of any of the embodiments described herein, whereinthe formulation has a pH of approximately 3-8 (e.g., approximately4-7.5, 5-8, 5-7.5, 5-6.5, 5-7.0, 5.5-8.0, 5.5-7.7, 5.5-6.5, 6-7.5, or6-7.0). In some embodiments, the formulation has a pH of approximately5.5-6.5 (e.g., 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5). In someembodiments, the formulation has a pH of approximately 6.0.

In various embodiments, the present invention includes stableformulations of any of the embodiments described herein, wherein theformulation is a liquid formulation. In various embodiments, the presentinvention includes stable formulation of any of the embodimentsdescribed herein, wherein the formulation is formulated as lyophilizeddry powder.

In some embodiments, the present invention includes a stable formulationfor intrathecal administration comprising an arylsulfatase A (ASA)protein at a concentration ranging from approximately 1-300 mg/ml, NaClat a concentration of approximately 154 mM, polysorbate 20 at aconcentration of approximately 0.005%, and a pH of approximately 6.0. Insome embodiments, the ASA protein is at a concentration of approximately10 mg/ml. In some embodiments, the ASA protein is at a concentration ofapproximately 30 mg/ml, 40 mg/ml, 50 mg/ml, 100 mg/ml, 150 mg/ml, 200mg/ml, 250 mg/ml, or 300 mg/ml.

In various aspects, the present invention includes a containercomprising a single dosage form of a stable formulation in variousembodiments described herein. In some embodiments, the container isselected from an ampule, a vial, a bottle, a cartridge, a reservoir, alyo-ject, or a pre-filled syringe. In some embodiments, the container isa pre-filled syringe. In some embodiments, the pre-filled syringe isselected from borosilicate glass syringes with baked silicone coating,borosilicate glass syringes with sprayed silicone, or plastic resinsyringes without silicone. In some embodiments, the stable formulationis present in a volume of less than about 50 mL (e.g., less than about45 ml, 40 ml, 35 ml, 30 ml, 25 ml, 20 ml, 15 ml, 10 ml, 5 ml, 4 ml, 3ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml). In some embodiments, thestable formulation is present in a volume of less than about 3.0 mL.

In various aspects, the present invention includes methods of treatingMetachromatic Leukodystrophy Disease including the step of administeringintrathecally to a subject in need of treatment a formulation accordingto any of the embodiments described herein.

In some embodiments, the present invention includes a method of treatingMetachromatic Leukodystrophy Disease including a step of administeringintrathecally to a subject in need of treatment a formulation comprisingan arylsulfatase A (ASA) protein at a concentration ranging fromapproximately 1-300 mg/ml, NaCl at a concentration of approximately 154mM, polysorbate 20 at a concentration of approximately 0.005%, and a pHof approximately 6.

In some embodiments, the intrathecal administration results in nosubstantial adverse effects (e.g., severe immune response) in thesubject. In some embodiments, the intrathecal administration results inno substantial adaptive T cell-mediated immune response in the subject.

In some embodiments, the intrathecal administration of the formulationresults in delivery of the arylsulfatase A protein to various targettissues in the brain, the spinal cord, and/or peripheral organs. In someembodiments, the intrathecal administration of the formulation resultsin delivery of the arylsulfatase A protein to target brain tissues. Insome embodiments, the brain target tissues comprise white matter and/orneurons in the gray matter. In some embodiments, the arylsulfatase Aprotein is delivered to neurons, glial cells, perivascular cells and/ormeningeal cells. In some embodiments, the arylsulfatase A protein isfurther delivered to the neurons in the spinal cord.

In some embodiments, the intrathecal administration of the formulationfurther results in systemic delivery of the ASA protein in peripheraltarget tissues. In some embodiments, the peripheral target tissues areselected from liver, kidney, spleen and/or heart.

In some embodiments, the intrathecal administration of the formulationresults in lysosomal localization in brain target tissues, spinal cordneurons and/or peripheral target tissues. In some embodiments, theintrathecal administration of the formulation results in reduction ofsulfatide storage in the brain target tissues, spinal cord neuronsand/or peripheral target tissues. In some embodiments, the sulfatidestorage is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 1-fold, 1.5-fold, or 2-fold as compared to a control (e.g., thepre-treatment GAG storage in the subject). In some embodiments, theintrathecal administration of the formulation results in reducedvacuolization in neurons (e.g., by at least 20%, 40%, 50%, 60%, 80%,90%, 1-fold, 1.5-fold, or 2-fold as compared to a control). In someembodiments, the neurons comprises Purkinje cells.

In some embodiments, the intrathecal administration of the formulationresults in increased ASA enzymatic activity in the brain target tissues,spinal cord neurons and/or peripheral target tissues. In someembodiments, the ASA enzymatic activity is increased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or10-fold as compared to a control (e.g., the pre-treatment endogenousenzymatic activity in the subject). In some embodiments, the increasedASA enzymatic activity is at least approximately 10 nmol/hr/mg, 20nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg,80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600nmol/hr/mg.

In some embodiments, the ASA enzymatic activity is increased in thelumbar region. In some embodiments, the increased ASA enzymatic activityin the lumbar region is at least approximately 2000 nmol/hr/mg, 3000nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, or 10,000 nmol/hr/mg.

In some embodiments, the intrathecal administration of the formulationresults in reduced intensity, severity, or frequency, or delayed onsetof at least one symptom or feature of MLD. In some embodiments, the atleast one symptom or feature of the MLD is cognitive impairment; whitematter lesions; dilated perivascular spaces in the brain parenchyma,ganglia, corpus callosum, and/or brainstem; atrophy; and/orventriculomegaly.

In some embodiments, the intrathecal administration takes place onceevery two weeks. In some embodiments, the intrathecal administrationtakes place once every month. In some embodiments, the intrathecaladministration takes place once every two months. In some embodiments,the intrathecal administration is used in conjunction with intravenousadministration. In some embodiments, the intravenous administration isno more frequent than once every week. In some embodiments, theintravenous administration is no more frequent than once every twoweeks. In some embodiments, the intravenous administration is no morefrequent than once every month. In some embodiments, the intravenousadministration is no more frequent than once every two months. Incertain embodiments, the intraveneous administration is more frequentthan monthly administration, such as twice weekly, weekly, every otherweek, or twice monthly.

In some embodiments, intraveneous and intrathecal administrations areperformed on the same day. In some embodiments, the intraveneous andintrathecal administrations are not performed within a certain amount oftime of each other, such as not within at least 2 days, within at least3 days, within at least 4 days, within at least 5 days, within at least6 days, within at least 7 days, or within at least one week. In someembodiments, intraveneous and intrathecal administrations are performedon an alternating schedule, such as alternating administrations weekly,every other week, twice monthly, or monthly. In some embodiments, anintrathecal administration replaces an intravenous administration in anadministration schedule, such as in a schedule of intraveneousadministration weekly, every other week, twice monthly, or monthly,every third or fourth or fifth administration in that schedule can bereplaced with an intrathecal administration in place of an intraveneousadministration.

In some embodiments, intraveneous and intrathecal administrations areperformed sequentially, such as performing intraveneous administrationsfirst (e.g., weekly, every other week, twice monthly, or monthly dosingfor two weeks, a month, two months, three months, four months, fivemonths, six months, a year or more) followed by IT administrations (e.g,weekly, every other week, twice monthly, or monthly dosing for more thantwo weeks, a month, two months, three months, four months, five months,six months, a year or more). In some embodiments, intrathecaladministrations are performed first (e.g., weekly, every other week,twice monthly, monthly, once every two months, once every three monthsdosing for two weeks, a month, two months, three months, four months,five months, six months, a year or more) followed by intraveneousadministrations (e.g, weekly, every other week, twice monthly, ormonthly dosing for more than two weeks, a month, two months, threemonths, four months, five months, six months, a year or more).

In some embodiments, the intrathecal administration is used in absenceof intravenous administration.

In some embodiments, the intrathecal administration is used in absenceof concurrent immunosuppressive therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates exemplary arylsulfatase A (rhASA) concentration datain serum after IV administration.

FIG. 2 illustrates exemplary rhASA concentration data in serum afterIT-lumbar administration.

FIG. 3 illustrates exemplary rhASA concentration in CSF after IVadministration.

FIG. 4 illustrates exemplary rhASA concentration in CSF after IT-lumbaradministration.

FIG. 5 illustrates exemplary analysis of the effect of buffer and pH onthe thermal stability of rhASA.

FIG. 6 illustrates exemplary SDS-PAGE (Coomassie) analysis of rhASAafter two weeks at 40±2° C.

FIG. 7 illustrates exemplary SDS-PAGE (Coomassie) analysis of rhASA inIT formulations after 3 months at 5 and 25° C.

FIG. 8 depicts exemplary rhASA drug substance and drug productappearance after 48 hours of stirring (Panel A) and shaking (Panel B).

FIG. 9 depicts exemplary rhASA drug product appearance (w/o P20) with(n=2) and without headspace (n=1) after stirring for 48 hours.

FIG. 10 illustrates exemplary data demonstrating the buffering capacityof rhASA drug substance compared to buffer control when titrated withhydrochloric acid.

FIG. 11 illustrates exemplary data demonstrating the buffering capacityof rhASA drug substance compared to a buffer control when titrated with1M sodium hydroxide.

FIG. 12 depicts exemplary rhASA samples in saline, pH 6.0 varying byconcentration.

FIG. 13 illustrates exemplary SEC-HPLC analysis of rhASA (pH 5.5 mobilephase) in 154 mM NaCl, pH 5.9.

FIG. 14 illustrates exemplary SEC-HPLC analysis of rhASA (pH 7.0 mobilephase) in 154 mM NaCl, pH 5.9.

FIG. 15 illustrates exemplary size exclusion profiles of baseline and 11month stability samples for rhASA in 154 mM NaCl, pH 5.

FIG. 16 depicts exemplary photo-micrographs of brain tissue, meninges,infiltrates (mid and high dose groups, both sexes) after treatment.

FIG. 17 depicts exemplary photo-micrographs of brain tissue, meninges,infiltrates (mid and high dose groups, both sexes) after treatment.

FIG. 18 depicts exemplary photo-micrographs of brain tissue,perivascular, infiltrates (mid dose males; high dose females) aftertreatment.

FIG. 19 depicts exemplary Alcian blue staining of spinal cord ofimmunotolerant MLD mice treated with rhASA depicts exemplary resultsillustrating sulfatide reduction as determined by Alcian blue stainingof the cervical spinal cord in animals that received intrathecalinjections of recombinant hASA at days 1, 8, 15 and 22 at doses of 520mg/kg brain weight or vehicle control. As demonstrated, treatment withintrathecally injected recombinant hASA resulted in reduction ofsulfatide accumulation in the cervical spinal cord.

FIG. 20 illustrates exemplary morphometry analysis of Alcian bluestained spinal cord sections from immunotolerant MLD mice treated withrhASA, including exemplary results illustrating optical density ofAlcian blue in total spinal cord (T-Spinal Cord), total gray matter(T-GM), lumbar gray matter (L-GM), cervical gray matter (C-GM), totalwhite matter (T-WM), lumbar white matter (L-WM), and cervical whitematter (C-WM) as determined by morphometry analysis. As demonstrated, astatistically significant reduction in Alcian blue staining was observedin animals treated with rhASA as compared to a vehicle control.

FIG. 21 depicts exemplary reduction of LAMP staining in white matter(fimbria) of immunotolerant MLD mice treated with rhASA depictsexemplary results illustrating LAMP-1 levels in fimbria as determined byimmunohistochemistry. Magnification=20×. As demonstrated, treatment withintrathecally injected rhASA resulted in reduction of LAMP-1 in thecerebral white matter.

FIG. 22 illustrates exemplary morphometry Analysis of LAMP staining ofbrain from immunotolerant MLD mice treated with rhASA depicts exemplaryresults illustrating LAMP-1 staining intensity in corpus collosum (CC),fimbria (F), cerebellar white matter (CB-WM) and brain stem (BS) ofanimals treated with 20 mg/kg intravenous rhASA, 300 mg/kg brain weightintrathecal rhASA, 520 mg/kg brain weight intravenous rhASA, or vehiclecontrol.

FIG. 23 illustrates exemplary concentration of rhASA in brain punches ofvehicle-dosed juvenile cynomolgus monkeys following EOW IT dosing for6-months-main necropsy.

FIG. 24 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT dosing of rhASA at 1.8mg/dose for 6-months—main necropsy.

FIG. 25 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT Dosing of rhASA at 6.0mg/dose for 6-months—main necropsy.

FIG. 26 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT dosing of rhASA at 18.6mg/dose for 6-months—main necropsy.

FIG. 27 illustrates exemplary concentration of ASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT dosing (PBS-control) for6-months—recovery necropsy.

FIG. 28 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT dosing of vehicle for6-months—recovery necropsy.

FIG. 29 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT dosing of rhASA at 1.8mg/dose for 6-months—recovery necropsy

FIG. 30 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus monkeys following EOW IT dosing of rhASA at 6.0mg/dose for 6 months—recovery necropsy

FIG. 31 illustrates exemplary concentration of rhASA in brain punches ofjuvenile cynomolgus following EOW IT dosing of rhASA at 18.6 mg/dose for6-months—recovery necropsy

FIG. 32 illustrates exemplary concentration of rhASA in selected punchesfrom surface of brain for device control, vehicle, 1.8 mg, 6.0 mg and18.6 mg treated animals. (male and female separate, device control datais from recovery necropsy, all other data from main necropsy).

FIG. 33 illustrates exemplary concentration of rhASA in selected punchesfrom deep white area of brain for device control, vehicle, 1.8 mg, 6.0mg and 18.6 mg treated animals. (male and female separate, devicecontrol data is from recovery necropsy, all other data from mainnecropsy).

FIG. 34 illustrates exemplary concentration of rhASA in selected punchesfrom deep grey area of brain for device control, vehicle, 1.8 mg, 6.0 mgand 18.6 mg treated animals. (male and female separate, device controldata is from recovery necropsy, all other data from main necropsy).

FIG. 35 illustrates exemplary concentration of rhASA in selected punchesfrom various regions in device control, vehicle, 1.8.mg, 6.0 mg and 18.6mg treated animals. (male and female combined, device control data isfrom recovery necropsy, all other data from main necropsy).

FIG. 36 illustrates exemplary concentration of rhASA in spinal cordsections of juvenile cynomolgus monkeys following EOW IT dosing for6-months—recovery necroscopy.

FIG. 37 illustrates exemplary concentration of rhASA in liver ofjuvenile cynomolgus monkeys following EOW IT dosing for6-months—recovery necroscopy.

FIG. 38 illustrates exemplary anatomical locations of certain brainpunches.

FIG. 39 illustrates exemplary anatomical locations of certain brainpunches.

FIG. 40 illustrates exemplary anatomical locations of certain brainpunches.

FIG. 41 illustrates exemplary anatomical locations of certain brainpunches.

FIG. 42 illustrates exemplary anatomical locations of certain brainpunches.

FIG. 43 illustrates exemplary anatomical locations of certain brainpunches.

FIG. 44A-G illustrate the concentration of recombinant humanarylsulfatase A (rhASA) in extracted tissue punches from the braintissues of adult and juvenile cynomolgus monkeys administered either avehicle, 1.8 mg rhASA or 18.6 mg rhASA. Each of FIG. 44A-G correspondsto a region of the brain tissue depicted in FIG. 39.

FIGS. 45A and B illustrate exemplary comparison of the concentrations ofrecombinant human arylsulfatase A (rhASA) detected in the deep whitematter (FIG. 45A) or in the deep grey matter (FIG. 45B) brain tissues ofadult and juvenile cynomolgus monkeys which were intrathecally (IT) orintracerebroventricularly (ICV) administered rhASA.

FIG. 46A illustrate concentrations of rhASA detected in several tissuepunches obtained from juvenile (<12 months of age) cynomolgus monkeysIT-administered an 18.6 or a 1.8 mg dose of recombinant humanarylsulfatase A (rhASA). As illustrated in both FIGS. 40A-B, theconcentration of rhASA delivered to the tissues were within, orotherwise exceeded the target therapeutic concentration of 2.5 mg/mgprotein. The anatomical regions of brain tissue which correspond to eachof the punch numbers depicted in FIG. 46A and FIG. 46B are the:subcortical white matter (1); periventricular white matter and deepwhite matter (2); subcortical white matter (3); subcortical white matter(4); internal capsule (5); internal capsule caudate nucleus (6); deepwhite matter (7); subcortical white matter and cortex (8); putamen (9);temporal subcortical white matter and cortex (10), deep grey matter(11), deep grey matter (12), frontal periventricular & subcortical (13);subcortical white matter, cortex superficial perifalxian (14); corpuscallosum and pericallosal subcortical white matter (15); deepsubcortical white matter (16); deep grey matter (17); deep grey matter(18); periventricular white matter (19); deep subcortical white matter(20); hippocampus (21); corpus callosum (22); deep white matter (23);subcortical white matter, occipital lobe (24); and cerebellar whitematter (25).

FIG. 47A illustrates the area of deep white matter tissue extracted froma cynomolgus monkey IT-administered 1.8 mg of rhASA. FIG. 47Billustrates immunostaining of the deep white matter tissue and revealeddistribution of rhASA in relevant cells. FIG. 47C illustrates that theIT-administered rhASA demonstrated organelle co-localization in the deepwhite matter tissues of the cynomolgus monkey and in particular in thelysosomes. In FIG. 47C, ASA immunostaining is illustrated in the topleft box.

FIG. 48 compares the distribution of ¹²⁴I-labeled arylsulfatase A(rhASA) using PET scanning 24 hours following either IT- orICV-administration of such labeled rhASA to a cynomolgus monkey.

FIG. 49 illustrates the distribution of ¹²⁴I-labeled ASA immediatelyfollowing ICV administration to a cynomolgus monkey, and compares thedistribution of IT-administered ¹²⁴I-labeled ASA within 2-5 hr. Asdemonstrated, IT administration delivered the ¹²⁴I-labeled ASA to thesame initial compartments (cisternae and proximal spine) as that shownfor the ICV administration.

FIG. 50 depicts exemplary ICV and IT administration in a mouse model.

FIG. 51 depicts an exemplary intrathecal drug delivery device (IDDD).

FIG. 52 depicts an exemplary PORT-A-CATH® low profile intrathecalimplantable access system.

FIG. 53 depicts an exemplary intrathecal drug delivery device (IDDD).

FIG. 54 depicts an exemplary intrathecal drug delivery device (IDDD),which allows for in-home administration for CNS enzyme replacementtherapy (ERT).

FIG. 55 illustrates and exemplary diagram of an intrathecal drugdelivery device (IDDD) with a securing mechanism.

FIG. 56 depicts exemplary locations within a patient's body where anIDDD may be placed; FIG. 56B depicts various components of anintrathecal drug delivery device (IDDD); and FIG. 56C depicts anexemplary insertion location within a patient's body for IT-lumbarinjection.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes increasing levels of relevant proteinor its activity that is deficient in relevant disease tissues.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Bulking agent: As used herein, the term “bulking agent” refers to acompound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake (e.g., facilitates theproduction of an essentially uniform lyophilized cake which maintains anopen pore structure). Exemplary bulking agents include mannitol,glycine, sodium chloride, hydroxyethyl starch, lactose, sucrose,trehalose, polyethylene glycol and dextran.

Cation-independent mannose-6-phosphate receptor (CI-MPR): As usedherein, the term “cation-independent mannose-6-phosphate receptor(CI-MPR)” refers to a cellular receptor that binds mannose-6-phosphate(M6P) tags on acid hydrolase precursors in the Golgi apparatus that aredestined for transport to the lysosome. In addition tomannose-6-phosphates, the CI-MPR also binds other proteins includingIGF-II. The CI-MPR is also known as “M6P/IGF-II receptor,”“CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor.” Theseterms and abbreviations thereof are used interchangeably herein.

Concurrent immunosuppressant therapy: As used herein, the term“concurrent immunosuppressant therapy” includes any immunosuppressanttherapy used as pre-treatment, preconditioning or in parallel to atreatment method.

Diluent: As used herein, the term “diluent” refers to a pharmaceuticallyacceptable (e.g., safe and non-toxic for administration to a human)diluting substance useful for the preparation of a reconstitutedformulation. Exemplary diluents include sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic protein forthe patient to be treated. Each unit contains a predetermined quantityof active material calculated to produce the desired therapeutic effect.It will be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Enzyme replacement therapy (ERT): As used herein, the term “enzymereplacement therapy (ERT)” refers to any therapeutic strategy thatcorrects an enzyme deficiency by providing the missing enzyme. In someembodiments, the missing enzyme is provided by intrathecaladministration. In some embodiments, the missing enzyme is provided byinfusing into bloodstream. Once administered, enzyme is taken up bycells and transported to the lysosome, where the enzyme acts toeliminate material that has accumulated in the lysosomes due to theenzyme deficiency. Typically, for lysosomal enzyme replacement therapyto be effective, the therapeutic enzyme is delivered to lysosomes in theappropriate cells in target tissues where the storage defect ismanifest.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from a disease.

Intrathecal administration: As used herein, the term “intrathecaladministration” or “intrathecal injection” refers to an injection intothe spinal canal (intrathecal space surrounding the spinal cord).Various techniques may be used including, without limitation, lateralcerebroventricular injection through a burrhole or cisternal or lumbarpuncture or the like. In some embodiments, “intrathecal administration”or “intrathecal delivery” according to the present invention refers toIT administration or delivery via the lumbar area or region, i.e.,lumbar IT administration or delivery. As used herein, the term “lumbarregion” or “lumbar area” refers to the area between the third and fourthlumbar (lower back) vertebrae and, more inclusively, the L2-S1 region ofthe spine.

Linker: As used herein, the term “linker” refers to, in a fusionprotein, an amino acid sequence other than that appearing at aparticular position in the natural protein and is generally designed tobe flexible or to interpose a structure, such as an a-helix, between twoprotein moieties. A linker is also referred to as a spacer.

Lyoprotectant: As used herein, the term “lyoprotectant” refers to amolecule that prevents or reduces chemical and/or physical instabilityof a protein or other substance upon lyophilization and subsequentstorage. Exemplary lyoprotectants include sugars such as sucrose ortrehalose; an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate:a polyol such as trihydric or higher sugar alcohols, e.g. glycerin,erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;propylene glycol; polyethylene glycol; Pluronics; and combinationsthereof. In some embodiments, a lyoprotectant is a non-reducing sugar,such as trehalose or sucrose.

Lysosomal enzyme: As used herein, the term “lysosomal enzyme” refers toany enzyme that is capable of reducing accumulated materials inmammalian lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms. Lysosomal enzymes suitable for theinvention include both wild-type or modified lysosomal enzymes and canbe produced using recombinant and synthetic methods or purified fromnature sources. Exemplary lysosomal enzymes are listed in Table 1.

Lysosomal enzyme deficiency: As used herein, “lysosomal enzymedeficiency” refers to a group of genetic disorders that result fromdeficiency in at least one of the enzymes that are required to breakmacromolecules (e.g., enzyme substrates) down to peptides, amino acids,monosaccharides, nucleic acids and fatty acids in lysosomes. As aresult, individuals suffering from lysosomal enzyme deficiencies haveaccumulated materials in various tissues (e.g., CNS, liver, spleen, gut,blood vessel walls and other organs).

Lysosomal Storage Disease: As used herein, the term “lysosomal storagedisease” refers to any disease resulting from the deficiency of one ormore lysosomal enzymes necessary for metabolizing naturalmacromolecules. These diseases typically result in the accumulation ofun-degraded molecules in the lysosomes, resulting in increased numbersof storage granules (also termed storage vesicles). These diseases andvarious examples are described in more detail below.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Replacement enzyme: As used herein, the term “replacement enzyme” refersto any enzyme that can act to replace at least in part the deficient ormissing enzyme in a disease to be treated. In some embodiments, the term“replacement enzyme” refers to any enzyme that can act to replace atleast in part the deficient or missing lysosomal enzyme in a lysosomalstorage disease to be treated. In some embodiments, a replacement enzymeis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Replacement enzymes suitable for the invention include bothwild-type or modified lysosomal enzymes and can be produced usingrecombinant and synthetic methods or purified from nature sources. Areplacement enzyme can be a recombinant, synthetic, gene-activated ornatural enzyme.

Soluble: As used herein, the term “soluble” refers to the ability of atherapeutic agent to form a homogenous solution. In some embodiments,the solubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of action(e.g., the cells and tissues of the brain) is sufficient to permit thedelivery of a therapeutically effective amount of the therapeutic agentto the targeted site of action. Several factors can impact thesolubility of the therapeutic agents. For example, relevant factorswhich may impact protein solubility include ionic strength, amino acidsequence and the presence of other co-solubilizing agents or salts(e.g., calcium salts). In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions. In some embodiments, therapeutic agents in accordancewith the present invention are soluble in its correspondingpharmaceutical composition. It will be appreciated that, while isotonicsolutions are generally preferred for parenterally administered drugs,the use of isotonic solutions may limit adequate solubility for sometherapeutic agents and, in particular some proteins and/or enzymes.Slightly hypertonic solutions (e.g., up to 175 mM sodium chloride in 5mM sodium phosphate at pH 7.0) and sugar-containing solutions (e.g., upto 2% sucrose in 5 mM sodium phosphate at pH 7.0) have been demonstratedto be well tolerated in monkeys. For example, the most common approvedCNS bolus formulation composition is saline (150 mM NaCl in water).

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). Ingeneral, pharmaceutical compositions described herein have beenformulated such that they are capable of stabilizing, or alternativelyslowing or preventing the degradation, of one or more therapeutic agentsformulated therewith (e.g., recombinant proteins). In the context of aformulation a stable formulation is one in which the therapeutic agenttherein essentially retains its physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization). For proteinstability, it can be measure by formation of high molecular weight (HMW)aggregates, loss of enzyme activity, generation of peptide fragments andshift of charge profiles.

Subject: As used herein, the term “subject” means any mammal, includinghumans. In certain embodiments of the present invention the subject isan adult, an adolescent or an infant. Also contemplated by the presentinvention are the administration of the pharmaceutical compositionsand/or performance of the methods of treatment in-utero.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Synthetic CSF: As used herein, the term “synthetic CSF” refers to asolution that has pH, electrolyte composition, glucose content andosmalarity consistent with the cerebrospinal fluid. Synthetic CSF isalso referred to as artificial CSF. In some embodiments, synthetic CSFis an Elliott's B solution.

Suitable for CNS delivery: As used herein, the phrase “suitable for CNSdelivery” or “suitable for intrathecal delivery” as it relates to thepharmaceutical compositions of the present invention generally refers tothe stability, tolerability, and solubility properties of suchcompositions, as well as the ability of such compositions to deliver aneffective amount of the therapeutic agent contained therein to thetargeted site of delivery (e.g., the CSF or the brain).

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by the lysosomal storage disease to be treatedor any tissue in which the deficient lysosomal enzyme is normallyexpressed. In some embodiments, target tissues include those tissues inwhich there is a detectable or abnormally high amount of enzymesubstrate, for example stored in the cellular lysosomes of the tissue,in patients suffering from or susceptible to the lysosomal storagedisease. In some embodiments, target tissues include those tissues thatdisplay disease-associated pathology, symptom, or feature. In someembodiments, target tissues include those tissues in which the deficientlysosomal enzyme is normally expressed at an elevated level. As usedherein, a target tissue may be a brain target tissue, a spinal cordtarget tissue an/or a peripheral target tissue. Exemplary target tissuesare described in detail below.

Therapeutic moiety: As used herein, the term “therapeutic moiety” refersto a portion of a molecule that renders the therapeutic effect of themolecule. In some embodiments, a therapeutic moiety is a polypeptidehaving therapeutic activity.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticprotein (e.g., replacement enzyme) which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). In particular, the “therapeuticallyeffective amount” refers to an amount of a therapeutic protein orcomposition effective to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect, such as by ameliorating symptoms associated with the disease,preventing or delaying the onset of the disease, and/or also lesseningthe severity or frequency of symptoms of the disease. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular therapeutic protein, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents.Also, the specific therapeutically effective amount (and/or unit dose)for any particular patient may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific pharmaceutical agent employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration,and/or rate of excretion or metabolism of the specific fusion proteinemployed; the duration of the treatment; and like factors as is wellknown in the medical arts.

Tolerable: As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic protein (e.g.,lysosomal enzyme) that partially or completely alleviates, ameliorates,relieves, inhibits, delays onset of, reduces severity of and/or reducesincidence of one or more symptoms or features of a particular disease,disorder, and/or condition (e.g., Hunters syndrome, Sanfilippo Bsyndrome). Such treatment may be of a subject who does not exhibit signsof the relevant disease, disorder and/or condition and/or of a subjectwho exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, such treatment may be of asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, improved methods andcompositions for effective direct delivery of a therapeutic agent to thecentral nervous system (CNS). As discussed above, the present inventionis based on unexpected discovery that a replacement enzyme (e.g., an ASAprotein) for a lysososmal storage disease (e.g., MetachromaticLeukodystrophy Disease) can be directly introduced into thecerebrospinal fluid (CSF) of a subject in need of treatment at a highconcentration without inducing substantial adverse effects in thesubject. More surprisingly, the present inventors found that thereplacement enzyme may be delivered in a simple saline or buffer-basedformulation, without using synthetic CSF. Even more unexpectedly,intrathecal delivery according to the present invention does not resultin substantial adverse effects, such as severe immune response, in thesubject. Therefore, in some embodiments, intrathecal delivery accordingto the present invention may be used in absence of concurrentimmunosuppressant therapy (e.g., without induction of immune toleranceby pre-treatment or pre-conditioning).

In some embodiments, intrathecal delivery according to the presentinvention permits efficient diffusion across various brain tissuesresulting in effective delivery of the replacement enzyme in varioustarget brain tissues in surface, shallow and/or deep brain regions. Insome embodiments, intrathecal delivery according to the presentinvention resulted in sufficient amount of replacement enzymes enteringthe peripheral circulation. As a result, in some cases, intrathecaldelivery according to the present invention resulted in delivery of thereplacement enzyme in peripheral tissues, such as liver, heart, spleenand kidney. This discovery is unexpected and can be particular usefulfor the treatment of lysosomal storage diseases that have both CNS andperipheral components, which would typically require both regularintrathecal administration and intravenous administration. It iscontemplated that intrathecal delivery according to the presentinvention may allow reduced dosing and/or frequency of iv injectionwithout compromising therapeutic effects in treating peripheralsymptoms.

The present invention provides various unexpected and beneficialfeatures that allow efficient and convenient delivery of replacementenzymes to various brain target tissues, resulting in effectivetreatment of lysosomal storage diseases that have CNS indications.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Therapeutic Proteins

In some embodiments, inventive methods and compositions provided by thepresent invention are used to deliver an arylsulfatase A (ASA) proteinto the CNS for treatment of Metachromatic Leukodystrophy Disease. Asuitable ASA protein can be any molecule or a portion of a molecule thatcan substitute for naturally-occurring arylsulfatase A (ASA) proteinactivity or rescue one or more phenotypes or symptoms associated withASA-deficiency. In some embodiments, a replacement enzyme suitable forthe invention is a polypeptide having an N-terminus and a C-terminus andan amino acid sequence substantially similar or identical to maturehuman ASA protein.

Typically, human ASA is produced as a precursor molecule that isprocessed to a mature form. This process generally occurs by removingthe 18 amino acid signal peptide. Typically, the precursor form is alsoreferred to as full-length precursor or full-length ASA protein, whichcontains 507 amino acids. The N-terminal 18 amino acids are cleaved,resulting in a mature form that is 489 amino acids in length. Thus, itis contemplated that the N-terminal 18 amino acids is generally notrequired for the ASA protein activity. The amino acid sequences of themature form (SEQ ID NO:1) and full-length precursor (SEQ ID NO:2) of atypical wild-type or naturally-occurring human ASA protein are shown inTable 1.

TABLE 1 Human Arylsulfatase A Mature FormRPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARYMAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTPRPACCHCPDPHA (SEQ ID NO: 1) Full-LengthMGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAA PrecursorGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARYMAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTPRPACCHCPDPHA (SEQ ID NO: 2)

Thus, in some embodiments, a therapeutic moiety suitable for the presentinvention is mature human ASA protein (SEQ ID NO:1). In someembodiments, a suitable therapeutic moiety may be a homologue or ananalogue of mature human ASA protein. For example, a homologue or ananalogue of mature human ASA protein may be a modified mature human ASAprotein containing one or more amino acid substitutions, deletions,and/or insertions as compared to a wild-type or naturally-occurring ASAprotein (e.g., SEQ ID NO:1), while retaining substantial ASA proteinactivity. Thus, in some embodiments, a therapeutic moiety suitable forthe present invention is substantially homologous to mature human ASAprotein (SEQ ID NO:1). In some embodiments, a therapeutic moietysuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more homologous to SEQ ID NO:1. In someembodiments, a therapeutic moiety suitable for the present invention issubstantially identical to mature human ASA protein (SEQ ID NO:1). Insome embodiments, a therapeutic moiety suitable for the presentinvention has an amino acid sequence at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:1. In some embodiments, a therapeutic moietysuitable for the present invention contains a fragment or a portion ofmature human ASA protein.

Alternatively, a replacement enzyme suitable for the present inventionis full-length ASA protein. In some embodiments, a suitable replacementenzyme may be a homologue or an analogue of full-length human ASAprotein. For example, a homologue or an analogue of full-length humanASA protein may be a modified full-length human ASA protein containingone or more amino acid substitutions, deletions, and/or insertions ascompared to a wild-type or naturally-occurring full-length ASA protein(e.g., SEQ ID NO:2), while retaining substantial ASA protein activity.Thus, In some embodiments, a replacement enzyme suitable for the presentinvention is substantially homologous to full-length human ASA protein(SEQ ID NO:2). In some embodiments, a replacement enzyme suitable forthe present invention has an amino acid sequence at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homologous to SEQ ID NO:2. In some embodiments, areplacement enzyme suitable for the present invention is substantiallyidentical to SEQ ID NO:2. In some embodiments, a replacement enzymesuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. In someembodiments, a replacement enzyme suitable for the present inventioncontains a fragment or a portion of full-length human ASA protein. Asused herein, a full-length ASA protein typically contains signal peptidesequence.

In some embodiments, a therapeutic protein includes a targeting moiety(e.g., a lysosome targeting sequence) and/or a membrane-penetratingpeptide. In some embodiments, a targeting sequence and/or amembrane-penetrating peptide is an intrinsic part of the therapeuticmoiety (e.g., via a chemical linkage, via a fusion protein). In someembodiments, a targeting sequence contains a mannose-6-phosphate moiety.In some embodiments, a targeting sequence contains an IGF-I moiety. Insome embodiments, a targeting sequence contains an IGF-II moiety.

Other Lysosomal Storage Diseases and Replacement Enzymes

It is contemplated that inventive methods and compositions according tothe present invention can be used to treat other lysosomal storagediseases, in particular those lysosomal storage diseases having CNSetiology and/or symptoms, including, but are not limited to,aspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, Danon disease, Fabry disease, Farberlipogranulomatosis, Farber disease, fucosidosis, galactosialidosis typesI/II, Gaucher disease types I/II/III, globoid cell leukodystrophy,Krabbe disease, glycogen storage disease II, Pompe disease,GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachsdisease, GM2-gangliosidosis type II, Sandhoff disease,GM2-gangliosidosis, α-mannosidosis types I/II, .beta.-mannosidosis,metachromatic leukodystrophy, mucolipidosis type I, sialidosis typesI/II, mucolipidosis types II/III, I-cell disease, mucolipidosis typeIIIC pseudo-Hurler polydystrophy, mucopolysaccharidosis type I,mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA,Sanfilippo syndrome, mucopolysaccharidosis type IIIB,mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosistype IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII,Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatasedeficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease, CLN2Batten disease, Niemann-Pick disease types A/B, Niemann-Pick diseasetype C1, Niemann-Pick disease type C2, pycnodysostosis, Schindlerdisease types I/II, Gaucher disease and sialic acid storage disease.

A detailed review of the genetic etiology, clinical manifestations, andmolecular biology of the lysosomal storage diseases are detailed inScriver et al., eds., The Metabolic and Molecular Basis of InheritedDisease, 7.sup.th Ed., Vol. II, McGraw Hill, (1995). Thus, the enzymesdeficient in the above diseases are known to those of skill in the art,some of these are exemplified in Table 2 below:

TABLE 2 Disease Name Enzyme Deficiency Substance Stored Pompe DiseaseAcid-a1, 4- Glycogen α1-4 Glucosidase linked Oligosaccharides GM1Gangliodsidosis β-Galactosidase GM₁ Gangliosides Tay-Sachs Diseaseβ-Hexosaminidase A GM₂ Ganglioside GM2 Gangliosidosis: GM₂ Activator GM₂Ganglioside AB Variant Protein Sandhoff Disease β-Hexosaminidase GM₂Ganglioside A&B Fabry Disease α-Galactosidase A Globosides GaucherDisease Glucocerebrosidase Glucosylceramide Metachromatic ArylsulfataseA Sulphatides Leukodystrophy Krabbe Disease GalactosylceramidaseGalactocerebroside Niemann Pick, Types Acid Sphingomyelin A & BSphingomyelinase Niemann-Pick, Type C Cholesterol SphingomyelinEsterification Defect Niemann-Pick, Type D Unknown Sphingomyelin FarberDisease Acid Ceramidase Ceramide Wolman Disease Acid Lipase CholesterylEsters Hurler Syndrome α-L-Iduronidase Heparan & (MPS IH) DermatanSulfates Scheie Syndrome α-L-Iduronidase Heparan & (MPS IS) Dermatan,Sulfates Hurler-Scheie α-L-Iduronidase Heparan & (MPS IH/S) DermatanSulfates Hunter Syndrome Iduronate Sulfatase Heparan & (MPS II) DermatanSulfates Sanfilippo A Heparan N-Sulfatase Heparan (MPS IIIA) SulfateSanfilippo B α-N-Acetyl- Heparan (MPS IIIB) glucosaminidase SulfateSanfilippo C Acetyl-CoA- Heparan (MPS IIIC) Glucosaminide SulfateAcetyltransferase Sanfilippo D N-Acetylglucosamine- Heparan (MPS HID)6-Sulfatase Sulfate Morquio B β-Galactosidase Keratan (MPS IVB) SulfateMaroteaux-Lamy Arylsulfatase B Dermatan (MPS VI) Sulfate Sly Syndromeβ-Glucuronidase (MPS VII) α-Mannosidosis α-Mannosidase Mannose/Oligosaccharides β-Mannosidosis β-Mannosidase Mannose/ OligosaccharidesFucosidosis α-L-Fucosidase Fucosyl Oligosaccharides Aspartyl-N-Aspartyl-β- Aspartylgluco- glucosaminuria Glucosaminidase samineAsparagines Sialidosis α-Neuraminidase Sialyloligo- (Mucolipidosis I)saccharides Galactosialidosis Lysosomal Protective Sialyloligo-(Goldberg Syndrome) Protein Deficiency saccharides Schindler Diseaseα-N-Acetyl- Galactosaminidase Mucolipidosis II (I-CellN-Acetylglucosamine- Heparan Sulfate Disease) 1-PhosphotransferaseMucolipidosis III Same as ML II (Pseudo-Hurler Polydystrophy) CystinosisCystine Transport Free Cystine Protein Salla Disease Sialic AcidTransport Free Sialic Acid and Protein Glucuronic Acid Infantile SialicAcid Sialic Acid Transport Free Sialic Acid and Storage Disease ProteinGlucuronic Acid Infantile Neuronal Palmitoyl-Protein Lipofuscins CeroidLipofuscinosis Thioesterase Mucolipidosis IV Unknown Gangliosides &Hyaluronic Acid Prosaposin Saposins A, B, C or D

Inventive methods according to the present invention may be used todeliver various other replacement enzymes. As used herein, replacementenzymes suitable for the present invention may include any enzyme thatcan act to replace at least partial activity of the deficient or missinglysosomal enzyme in a lysosomal storage disease to be treated. In someembodiments, a replacement enzyme is capable of reducing accumulatedsubstance in lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms.

In some embodiments, a suitable replacement enzyme may be any lysosomalenzyme known to be associated with the lysosomal storage disease to betreated. In some embodiments, a suitable replacement enzyme is an enzymeselected from the enzyme listed in Table 2 above.

In some embodiments, a replacement enzyme suitable for the invention mayhave a wild-type or naturally occurring sequence. In some embodiments, areplacement enzyme suitable for the invention may have a modifiedsequence having substantial homology or identify to the wild-type ornaturally-occurring sequence (e.g., having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to the wild-type ornaturally-occurring sequence).

A replacement enzyme suitable for the present invention may be producedby any available means. For example, replacement enzymes may berecombinantly produced by utilizing a host cell system engineered toexpress a replacement enzyme-encoding nucleic acid. Alternatively oradditionally, replacement enzymes may be produced by activatingendogenous genes. Alternatively or additionally, replacement enzymes maybe partially or fully prepared by chemical synthesis. Alternatively oradditionally, replacements enzymes may also be purified from naturalsources.

Where enzymes are recombinantly produced, any expression system can beused. To give but a few examples, known expression systems include, forexample, egg, baculovirus, plant, yeast, or mammalian cells.

In some embodiments, enzymes suitable for the present invention areproduced in mammalian cells. Non-limiting examples of mammalian cellsthat may be used in accordance with the present invention include BALB/cmouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts(PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g.,HT1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

In some embodiments, inventive methods according to the presentinvention are used to deliver replacement enzymes produced from humancells. In some embodiments, inventive methods according to the presentinvention are used to deliver replacement enzymes produced from CHOcells.

In some embodiments, replacement enzymes delivered using a method of theinvention contain a moiety that binds to a receptor on the surface ofbrain cells to facilitate cellular uptake and/or lysosomal targeting.For example, such a receptor may be the cation-independentmannose-6-phosphate receptor (CI-MPR) which binds themannose-6-phosphate (M6P) residues. In addition, the CI-MPR also bindsother proteins including IGF-II. In some embodiments, a replacementenzyme suitable for the present invention contains M6P residues on thesurface of the protein. In some embodiments, a replacement enzymesuitable for the present invention may contain bis-phosphorylatedoligosaccharides which have higher binding affinity to the CI-MPR. Insome embodiments, a suitable enzyme contains up to about an average ofabout at least 20% bis-phosphorylated oligosaccharides per enzyme. Inother embodiments, a suitable enzyme may contain about 10%, 15%, 18%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bis-phosphorylatedoligosaccharides per enzyme. While such bis-phosphorylatedoligosaccharides may be naturally present on the enzyme, it should benoted that the enzymes may be modified to possess such oligosaccharides.For example, suitable replacement enzymes may be modified by certainenzymes which are capable of catalyzing the transfer ofN-acetylglucosamine-L-phosphate from UDP-GlcNAc to the 6′ position ofα-1,2-linked mannoses on lysosomal enzymes. Methods and compositions forproducing and using such enzymes are described by, for example, Canfieldet al. in U.S. Pat. No. 6,537,785, and U.S. Pat. No. 6,534,300, eachincorporated herein by reference.

In some embodiments, replacement enzymes for use in the presentinvention may be conjugated or fused to a lysosomal targeting moietythat is capable of binding to a receptor on the surface of brain cells.A suitable lysosomal targeting moiety can be IGF-I, IGF-II, RAP, p97,and variants, homologues or fragments thereof (e.g., including thosepeptide having a sequence at least 70%, 75%, 80%, 85%, 90%, or 95%identical to a wild-type mature human IGF-I, IGF-II, RAP, p97 peptidesequence).

In some embodiments, replacement enzymes suitable for the presentinvention have not been modified to enhance delivery or transport ofsuch agents across the BBB and into the CNS.

In some embodiments, a therapeutic protein includes a targeting moiety(e.g., a lysosome targeting sequence) and/or a membrane-penetratingpeptide. In some embodiments, a targeting sequence and/or amembrane-penetrating peptide is an intrinsic part of the therapeuticmoiety (e.g., via a chemical linkage, via a fusion protein). In someembodiments, a targeting sequence contains a mannose-6-phosphate moiety.In some embodiments, a targeting sequence contains an IGF-I moiety. Insome embodiments, a targeting sequence contains an IGF-II moiety.

Formulations

Aqueous pharmaceutical solutions and compositions (i.e., formulations)that are traditionally used to deliver therapeutic agents to the CNS ofa subject include unbuffered isotonic saline and Elliott's B solution,which is artificial CSF. A comparison depicting the compositions of CSFrelative to Elliott's B solution is included in Table 3 below. As shownin T Table 3, the concentration of Elliot's B Solution closely parallelsthat of the CSF. Elliott's B Solution, however contains a very lowbuffer concentration and accordingly may not provide the adequatebuffering capacity needed to stabilize therapeutic agents (e.g.,proteins), especially over extended periods of time (e.g., duringstorage conditions). Furthermore, Elliott's B Solution contains certainsalts which may be incompatible with the formulations intended todeliver some therapeutic agents, and in particular proteins or enzymes.For example, the calcium salts present in Elliott's B Solution arecapable of mediating protein precipitation and thereby reducing thestability of the formulation.

TABLE 3 Na⁺ K⁺ Ca⁺⁺ Mg⁺⁺ HCO3⁻ Cl⁻ Phosphorous Glucose Solution mEq/LmEq/L mEq/L mEq/L mEq/L mEq/L pH mg/L mg/L CSF 117-137 2.3 2.2 2.2 22.9113-127 7.31 1.2-2.1 45-80 Elliott's 149 2.6 2.7 2.4 22.6 132 6.0-7.52.3 80 B Sol'n

The present invention provides formulations, in either aqueous,pre-lyophilized, lyophilized or reconstituted form, for therapeuticagents that have been formulated such that they are capable ofstabilizing, or alternatively slowing or preventing the degradation, ofone or more therapeutic agents formulated therewith (e.g., recombinantproteins). In some embodiments, the present formulations providelyophilization formulation for therapeutic agents. In some embodiments,the present formulations provide aqueous formulations for therapeuticagents. In some embodiments the formulations are stable formulations.

Stable Formulations

As used herein, the term “stable” refers to the ability of thetherapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., preferably for at least 1, 3, 6, 12, 18, 24, 30, 36 months ormore). In the context of a formulation a stable formulation is one inwhich the therapeutic agent therein essentially retains its physicaland/or chemical integrity and biological activity upon storage andduring processes (such as freeze/thaw, mechanical mixing andlyophilization). For protein stability, it can be measure by formationof high molecular weight (HMW) aggregates, loss of enzyme activity,generation of peptide fragments and shift of charge profiles.

Stability of the therapeutic agent is of particular importance withrespect to the maintenance of the specified range of the therapeuticagent concentration required to enable the agent to serve its intendedtherapeutic function. Stability of the therapeutic agent may be furtherassessed relative to the biological activity or physiochemical integrityof the therapeutic agent over extended periods of time. For example,stability at a given time point may be compared against stability at anearlier time point (e.g., upon formulation day 0) or againstunformulated therapeutic agent and the results of this comparisonexpressed as a percentage. Preferably, the pharmaceutical compositionsof the present invention maintain at least 100%, at least 99%, at least98%, at least 97% at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 65%, at least 60%, at least55% or at least 50% of the therapeutic agent's biological activity orphysiochemical integrity over an extended period of time (e.g., asmeasured over at least about 6-12 months, at room temperature or underaccelerated storage conditions).

The therapeutic agents are preferably soluble in the pharmaceuticalcompositions of the present invention. The term “soluble” as it relatesto the therapeutic agents of the present invention refer to the abilityof such therapeutic agents to form a homogenous solution. Preferably thesolubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of action(e.g., the cells and tissues of the brain) is sufficient to permit thedelivery of a therapeutically effective amount of the therapeutic agentto the targeted site of action. Several factors can impact thesolubility of the therapeutic agents. For example, relevant factorswhich may impact protein solubility include ionic strength, amino acidsequence and the presence of other co-solubilizing agents or salts(e.g., calcium salts.) In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions.

Suitable formulations, in either aqueous, pre-lyophilized, lyophilizedor reconstituted form, may contain a therapeutic agent of interest atvarious concentrations. In some embodiments, formulations may contain aprotein or therapeutic agent of interest at a concentration in the rangeof about 0.1 mg/ml to 100 mg/ml (e.g., about 0.1 mg/ml to 80 mg/ml,about 0.1 mg/ml to 60 mg/ml, about 0.1 mg/ml to 50 mg/ml, about 0.1mg/ml to 40 mg/ml, about 0.1 mg/ml to 30 mg/ml, about 0.1 mg/ml to 25mg/ml, about 0.1 mg/ml to 20 mg/ml, about 0.1 mg/ml to 60 mg/ml, about0.1 mg/ml to 50 mg/ml, about 0.1 mg/ml to 40 mg/ml, about 0.1 mg/ml to30 mg/ml, about 0.1 mg/ml to 25 mg/ml, about 0.1 mg/ml to 20 mg/ml,about 0.1 mg/ml to 15 mg/ml, about 0.1 mg/ml to 10 mg/ml, about 0.1mg/ml to 5 mg/ml, about 1 mg/ml to 10 mg/ml, about 1 mg/ml to 20 mg/ml,about 1 mg/ml to 40 mg/ml, about 5 mg/ml to 100 mg/ml, about 5 mg/ml to50 mg/ml, or about 5 mg/ml to 25 mg/ml). In some embodiments,formulations according to the invention may contain a therapeutic agentat a concentration of approximately 1 mg/ml, 5 mg/ml, 10 mg/ml, 15mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml.

The formulations of the present invention are characterized by theirtolerability either as aqueous solutions or as reconstituted lyophilizedsolutions. As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Many therapeutic agents, and in particular the proteins and enzymes ofthe present invention, require controlled pH and specific excipients tomaintain their solubility and stability in the pharmaceuticalcompositions of the present invention. Table 4 below identifies typicalaspects of protein formulations considered to maintain the solubilityand stability of the protein therapeutic agents of the presentinvention.

TABLE 4 Parameter Typical Range/Type Rationale pH 5 to 7.5 For stabilitySometimes also for solubility Buffer type acetate, succinate, citrate,To maintain optimal pH histidine, phosphate or Tris May also affectstability Buffer 5-50 mM To maintain pH concentration May also stabilizeor add ionic strength Tonicifier NaCl, sugars, mannitol To renderiso-osmotic or isotonic solutions Surfactant Polysorbate 20, Tostabilize against interfaces polysorbate 80 and shear Other Amino acids(e.g. arginine) For enhanced solubility or at tens to hundreds of mMstability

Buffers

The pH of the formulation is an additional factor which is capable ofaltering the solubility of a therapeutic agent (e.g., an enzyme orprotein) in an aqueous formulation or for a pre-lyophilizationformulation. Accordingly the formulations of the present inventionpreferably comprise one or more buffers. In some embodiments the aqueousformulations comprise an amount of buffer sufficient to maintain theoptimal pH of said composition between about 4.0-8.0 (e.g., about 4.0,4.5, 5.0, 5.5, 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.5, or 8.0). In someembodiments, the pH of the formulation is between about 5.0-7.5, betweenabout 5.5-7.0, between about 6.0-7.0, between about 5.5-6.0, betweenabout 5.5-6.5, between about 5.0-6.0, between about 5.0-6.5 and betweenabout 6.0-7.5. Suitable buffers include, for example acetate, citrate,histidine, phosphate, succinate, tris(hydroxymethyl)aminomethane(“Tris”) and other organic acids. The buffer concentration and pH rangeof the pharmaceutical compositions of the present invention are factorsin controlling or adjusting the tolerability of the formulation. In someembodiments, a buffering agent is present at a concentration rangingbetween about 1 mM to about 150 mM, or between about 10 mM to about 50mM, or between about 15 mM to about 50 mM, or between about 20 mM toabout 50 mM, or between about 25 mM to about 50 mM. In some embodiments,a suitable buffering agent is present at a concentration ofapproximately 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40mM, 45 mM 50 mM, 75 mM, 100 mM, 125 mM or 150 mM.

Tonicity

In some embodiments, formulations, in either aqueous, pre-lyophilized,lyophilized or reconstituted form, contain an isotonicity agent to keepthe formulations isotonic. Typically, by “isotonic” is meant that theformulation of interest has essentially the same osmotic pressure ashuman blood. Isotonic formulations will generally have an osmoticpressure from about 240 mOsm/kg to about 350 mOsm/kg. Isotonicity can bemeasured using, for example, a vapor pressure or freezing point typeosmometers. Exemplary isotonicity agents include, but are not limitedto, glycine, sorbitol, mannitol, sodium chloride and arginine. In someembodiments, suitable isotonic agents may be present in aqueous and/orpre-lyophilized formulations at a concentration from about 0.01-5%(e.g., 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0,2.5, 3.0, 4.0 or 5.0%) by weight. In some embodiments, formulations forlyophilization contain an isotonicity agent to keep thepre-lyophilization formulations or the reconstituted formulationsisotonic.

While generally isotonic solutions are preferred for parenterallyadministered drugs, the use of isotonic solutions may change solubilityfor some therapeutic agents and in particular some proteins and/orenzymes. Slightly hypertonic solutions (e.g., up to 175 mM sodiumchloride in 5 mM sodium phosphate at pH 7.0) and sugar-containingsolutions (e.g., up to 2% sucrose in 5 mM sodium phosphate at pH 7.0)have been demonstrated to be well tolerated. The most common approvedCNS bolus formulation composition is saline (about 150 mM NaCl inwater).

Stabilizing Agents

In some embodiments, formulations may contain a stabilizing agent, orlyoprotectant, to protect the protein. Typically, a suitable stabilizingagent is a sugar, a non-reducing sugar and/or an amino acid. Exemplarysugars include, but are not limited to, dextran, lactose, mannitol,mannose, sorbitol, raffinose, sucrose and trehalose. Exemplary aminoacids include, but are not limited to, arginine, glycine and methionine.Additional stabilizing agents may include sodium chloride, hydroxyethylstarch and polyvinylpyrolidone. The amount of stabilizing agent in thelyophilized formulation is generally such that the formulation will beisotonic. However, hypertonic reconstituted formulations may also besuitable. In addition, the amount of stabilizing agent must not be toolow such that an unacceptable amount of degradation/aggregation of thetherapeutic agent occurs. Exemplary stabilizing agent concentrations inthe formulation may range from about 1 mM to about 400 mM (e.g., fromabout 30 mM to about 300 mM, and from about 50 mM to about 100 mM), oralternatively, from 0.1% to 15% (e.g., from 1% to 10%, from 5% to 15%,from 5% to 10%) by weight. In some embodiments, the ratio of the massamount of the stabilizing agent and the therapeutic agent is about 1:1.In other embodiments, the ratio of the mass amount of the stabilizingagent and the therapeutic agent can be about 0.1:1, 0.2:1, 0.25:1,0.4:1, 0.5:1, 1:1, 2:1, 2.6:1, 3:1, 4:1, 5:1, 10:1, or 20:1. In someembodiments, suitable for lyophilization, the stabilizing agent is alsoa lyoprotectant.

In some embodiments, liquid formulations suitable for the presentinvention contain amorphous materials. In some embodiments, liquidformulations suitable for the present invention contain a substantialamount of amorphous materials (e.g., sucrose-based formulations). Insome embodiments, liquid formulations suitable for the present inventioncontain partly crystalline/partly amorphous materials.

Bulking Agents

In some embodiments, suitable formulations for lyophilization mayfurther include one or more bulking agents. A “bulking agent” is acompound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake. For example, a bulkingagent may improve the appearance of lyophilized cake (e.g., essentiallyuniform lyophilized cake). Suitable bulking agents include, but are notlimited to, sodium chloride, lactose, mannitol, glycine, sucrose,trehalose, hydroxyethyl starch. Exemplary concentrations of bulkingagents are from about 1% to about 10% (e.g., 1.0%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%,9.0%, 9.5%, and 10.0%).

Surfactants

In some embodiments, it is desirable to add a surfactant toformulations. Exemplary surfactants include nonionic surfactants such asPolysorbates (e.g., Polysorbates 20 or 80); poloxamers (e.g., poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68, etc). Typically,the amount of surfactant added is such that it reduces aggregation ofthe protein and minimizes the formation of particulates oreffervescences. For example, a surfactant may be present in aformulation at a concentration from about 0.001-0.5% (e.g., about0.005-0.05%, or 0.005-0.01%). In particular, a surfactant may be presentin a formulation at a concentration of approximately 0.005%, 0.01%,0.02%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc. Alternatively, or inaddition, the surfactant may be added to the lyophilized formulation,pre-lyophilized formulation and/or the reconstituted formulation.

Other pharmaceutically acceptable carriers, excipients or stabilizerssuch as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the formulation (and/orthe lyophilized formulation and/or the reconstituted formulation)provided that they do not adversely affect the desired characteristicsof the formulation. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed andinclude, but are not limited to, additional buffering agents;preservatives; co-solvents; antioxidants including ascorbic acid andmethionine; chelating agents such as EDTA; metal complexes (e.g.,Zn-protein complexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

Formulations, in either aqueous, pre-lyophilized, lyophilized orreconstituted form, in accordance with the present invention can beassessed based on product quality analysis, reconstitution time (iflyophilized), quality of reconstitution (if lyophilized), high molecularweight, moisture, and glass transition temperature. Typically, proteinquality and product analysis include product degradation rate analysisusing methods including, but not limited to, size exclusion HPLC(SE-HPLC), cation exchange-HPLC (CEX-HPLC), X-ray diffraction (XRD),modulated differential scanning calorimetry (mDSC), reversed phase HPLC(RP-HPLC), multi-angle light scattering (MALS), fluorescence,ultraviolet absorption, nephelometry, capillary electrophoresis (CE),SDS-PAGE, and combinations thereof. In some embodiments, evaluation ofproduct in accordance with the present invention may include a step ofevaluating appearance (either liquid or cake appearance).

Generally, formulations (lyophilized or aqueous) can be stored forextended periods of time at room temperature. Storage temperature maytypically range from 0° C. to 45° C. (e.g., 4° C., 20° C., 25° C., 45°C. etc.). Formulations may be stored for a period of months to a periodof years. Storage time generally will be 24 months, 12 months, 6 months,4.5 months, 3 months, 2 months or 1 month. Formulations can be storeddirectly in the container used for administration, eliminating transfersteps.

Formulations can be stored directly in the lyophilization container (iflyophilized), which may also function as the reconstitution vessel,eliminating transfer steps. Alternatively, lyophilized productformulations may be measured into smaller increments for storage.Storage should generally avoid circumstances that lead to degradation ofthe proteins, including but not limited to exposure to sunlight, UVradiation, other forms of electromagnetic radiation, excessive heat orcold, rapid thermal shock, and mechanical shock.

Lyophilization

Inventive methods in accordance with the present invention can beutilized to lyophilize any materials, in particular, therapeutic agents.Typically, a pre-lyophilization formulation further contains anappropriate choice of excipients or other components such asstabilizers, buffering agents, bulking agents, and surfactants toprevent compound of interest from degradation (e.g., proteinaggregation, deamidation, and/or oxidation) during freeze-drying andstorage. The formulation for lyophilization can include one or moreadditional ingredients including lyoprotectants or stabilizing agents,buffers, bulking agents, isotonicity agents and surfactants.

After the substance of interest and any additional components are mixedtogether, the formulation is lyophilized. Lyophilization generallyincludes three main stages: freezing, primary drying and secondarydrying. Freezing is necessary to convert water to ice or some amorphousformulation components to the crystalline form. Primary drying is theprocess step when ice is removed from the frozen product by directsublimation at low pressure and temperature. Secondary drying is theprocess step when bounded water is removed from the product matrixutilizing the diffusion of residual water to the evaporation surface.Product temperature during secondary drying is normally higher thanduring primary drying. See, Tang X. et al. (2004) “Design offreeze-drying processes for pharmaceuticals: Practical advice,” Pharm.Res., 21:191-200; Nail S. L. et al. (2002) “Fundamentals offreeze-drying,” in Development and manufacture of proteinpharmaceuticals. Nail S. L. editor New York: Kluwer Academic/PlenumPublishers, pp 281-353; Wang et al. (2000) “Lyophilization anddevelopment of solid protein pharmaceuticals,” Int. J. Pharm., 203:1-60;Williams N. A. et al. (1984) “The lyophilization of pharmaceuticals; Aliterature review.” J. Parenteral Sci. Technol., 38:48-59. Generally,any lyophilization process can be used in connection with the presentinvention.

In some embodiments, an annealing step may be introduced during theinitial freezing of the product. The annealing step may reduce theoverall cycle time. Without wishing to be bound by any theories, it iscontemplated that the annealing step can help promote excipientcrystallization and formation of larger ice crystals due tore-crystallization of small crystals formed during supercooling, which,in turn, improves reconstitution. Typically, an annealing step includesan interval or oscillation in the temperature during freezing. Forexample, the freeze temperature may be −40° C., and the annealing stepwill increase the temperature to, for example, −10° C. and maintain thistemperature for a set period of time. The annealing step time may rangefrom 0.5 hours to 8 hours (e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 6, and 8hours). The annealing temperature may be between the freezingtemperature and 0° C.

Lyophilization may be performed in a container, such as a tube, a bag, abottle, a tray, a vial (e.g., a glass vial), syringe or any othersuitable containers. The containers may be disposable. Lyophilizationmay also be performed in a large scale or small scale. In someinstances, it may be desirable to lyophilize the protein formulation inthe container in which reconstitution of the protein is to be carriedout in order to avoid a transfer step. The container in this instancemay, for example, be a 3, 4, 5, 10, 20, 50 or 100 cc vial.

Many different freeze-dryers are available for this purpose such as Hullpilot scale dryer (SP Industries, USA), Genesis (SP Industries)laboratory freeze-dryers, or any freeze-dryers capable of controllingthe given lyophilization process parameters. Freeze-drying isaccomplished by freezing the formulation and subsequently subliming icefrom the frozen content at a temperature suitable for primary drying.Initial freezing brings the formulation to a temperature below about−20° C. (e.g., −50° C., −45° C., −40° C., −35° C., −30° C., −25° C.,etc.) in typically not more than about 4 hours (e.g., not more thanabout 3 hours, not more than about 2.5 hours, not more than about 2hours). Under this condition, the product temperature is typically belowthe eutectic point or the collapse temperature of the formulation.Typically, the shelf temperature for the primary drying will range fromabout −30 to 25° C. (provided the product remains below the meltingpoint during primary drying) at a suitable pressure, ranging typicallyfrom about 20 to 250 mTorr. The formulation, size and type of thecontainer holding the sample (e.g., glass vial) and the volume of liquidwill mainly dictate the time required for drying, which can range from afew hours to several days. A secondary drying stage is carried out atabout 0-60° C., depending primarily on the type and size of containerand the type of therapeutic protein employed. Again, volume of liquidwill mainly dictate the time required for drying, which can range from afew hours to several days.

As a general proposition, lyophilization will result in a lyophilizedformulation in which the moisture content thereof is less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, and less than about 0.5%.

Reconstitution

While the pharmaceutical compositions of the present invention aregenerally in an aqueous form upon administration to a subject, in someembodiments the pharmaceutical compositions of the present invention arelyophilized. Such compositions must be reconstituted by adding one ormore diluents thereto prior to administration to a subject. At thedesired stage, typically at an appropriate time prior to administrationto the patient, the lyophilized formulation may be reconstituted with adiluent such that the protein concentration in the reconstitutedformulation is desirable.

Various diluents may be used in accordance with the present invention.In some embodiments, a suitable diluent for reconstitution is water. Thewater used as the diluent can be treated in a variety of ways includingreverse osmosis, distillation, deionization, filtrations (e.g.,activated carbon, microfiltration, nanofiltration) and combinations ofthese treatment methods. In general, the water should be suitable forinjection including, but not limited to, sterile water or bacteriostaticwater for injection.

Additional exemplary diluents include a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Elliot's solution,Ringer's solution or dextrose solution. Suitable diluents may optionallycontain a preservative. Exemplary preservatives include aromaticalcohols such as benzyl or phenol alcohol. The amount of preservativeemployed is determined by assessing different preservativeconcentrations for compatibility with the protein and preservativeefficacy testing. For example, if the preservative is an aromaticalcohol (such as benzyl alcohol), it can be present in an amount fromabout 0.1-2.0%, from about 0.5-1.5%, or about 1.0-1.2%.

Diluents suitable for the invention may include a variety of additives,including, but not limited to, pH buffering agents, (e.g. Tris,histidine), salts (e.g., sodium chloride) and other additives (e.g.,sucrose) including those described above (e.g. stabilizing agents,isotonicity agents).

According to the present invention, a lyophilized substance (e.g.,protein) can be reconstituted to a concentration of at least 25 mg/ml(e.g., at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/) and inany ranges therebetween. In some embodiments, a lyophilized substance(e.g., protein) may be reconstituted to a concentration ranging fromabout 1 mg/ml to 100 mg/ml (e.g., from about 1 mg/ml to 50 mg/ml, from 1mg/ml to 100 mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1mg/ml to about 10 mg/ml, from about 1 mg/ml to about 25 mg/ml, fromabout 1 mg/ml to about 75 mg/ml, from about 10 mg/ml to about 30 mg/ml,from about 10 mg/ml to about 50 mg/ml, from about 10 mg/ml to about 75mg/ml, from about 10 mg/ml to about 100 mg/ml, from about 25 mg/ml toabout 50 mg/ml, from about 25 mg/ml to about 75 mg/ml, from about 25mg/ml to about 100 mg/ml, from about 50 mg/ml to about 75 mg/ml, fromabout 50 mg/ml to about 100 mg/ml). In some embodiments, theconcentration of protein in the reconstituted formulation may be higherthan the concentration in the pre-lyophilization formulation. Highprotein concentrations in the reconstituted formulation are consideredto be particularly useful where subcutaneous or intramuscular deliveryof the reconstituted formulation is intended. In some embodiments, theprotein concentration in the reconstituted formulation may be about 2-50times (e.g., about 2-20, about 2-10 times, or about 2-5 times) of thepre-lyophilized formulation. In some embodiments, the proteinconcentration in the reconstituted formulation may be at least about 2times (e.g., at least about 3, 4, 5, 10, 20, 40 times) of thepre-lyophilized formulation.

Reconstitution according to the present invention may be performed inany container. Exemplary containers suitable for the invention include,but are not limited to, such as tubes, vials, syringes (e.g.,single-chamber or dual-chamber), bags, bottles, and trays. Suitablecontainers may be made of any materials such as glass, plastics, metal.The containers may be disposable or reusable. Reconstitution may also beperformed in a large scale or small scale.

In some instances, it may be desirable to lyophilize the proteinformulation in the container in which reconstitution of the protein isto be carried out in order to avoid a transfer step. The container inthis instance may, for example, be a 3, 4, 5, 10, 20, 50 or 100 cc vial.In some embodiments, a suitable container for lyophilization andreconstitution is a dual chamber syringe (e.g., Lyo-Ject,® (Vetter)syringes). For example, a dual chamber syringe may contain both thelyophilized substance and the diluent, each in a separate chamber,separated by a stopper (see Example 5). To reconstitute, a plunger canbe attached to the stopper at the diluent side and pressed to movediluent into the product chamber so that the diluent can contact thelyophilized substance and reconstitution may take place as describedherein (see Example 5).

The pharmaceutical compositions, formulations and related methods of theinvention are useful for delivering a variety of therapeutic agents tothe CNS of a subject (e.g., intrathecally, intraventricularly orintracisternally) and for the treatment of the associated diseases. Thepharmaceutical compositions of the present invention are particularlyuseful for delivering proteins and enzymes (e.g., enzyme replacementtherapy) to subjects suffering from lysosomal storage disorders. Thelysosomal storage diseases represent a group of relatively rareinherited metabolic disorders that result from defects in lysosomalfunction. The lysosomal diseases are characterized by the accumulationof undigested macromolecules within the lysosomes, which results in anincrease in the size and number of such lysosomes and ultimately incellular dysfunction and clinical abnormalities.

CNS Delivery

It is contemplated that various stable formulations described herein aregenerally suitable for CNS delivery of therapeutic agents. Stableformulations according to the present invention can be used for CNSdelivery via various techniques and routes including, but not limitedto, intraparenchymal, intracerebral, intravetricular cerebral (ICV),intrathecal (e.g., IT-Lumbar, IT-cisterna magna) administrations and anyother techniques and routes for injection directly or indirectly to theCNS and/or CSF.

Intrathecal Delivery

In some embodiments, a replacement enzyme is delivered to the CNS in aformulation described herein. In some embodiments, a replacement enzymeis delivered to the CNS by administering into the cerebrospinal fluid(CSF) of a subject in need of treatment. In some embodiments,intrathecal administration is used to deliver a desired replacementenzyme (e.g., an ASA protein) into the CSF. As used herein, intrathecaladministration (also referred to as intrathecal injection) refers to aninjection into the spinal canal (intrathecal space surrounding thespinal cord). Various techniques may be used including, withoutlimitation, lateral cerebroventricular injection through a burrhole orcistemal or lumbar puncture or the like. Exemplary methods are describedin Lazorthes et al. Advances in Drug Delivery Systems and Applicationsin Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:169-179, the contents of which are incorporated herein by reference.

According to the present invention, an enzyme may be injected at anyregion surrounding the spinal canal. In some embodiments, an enzyme isinjected into the lumbar area or the cisterna magna orintraventricularly into a cerebral ventricle space. As used herein, theterm “lumbar region” or “lumbar area” refers to the area between thethird and fourth lumbar (lower back) vertebrae and, more inclusively,the L2-S1 region of the spine. Typically, intrathecal injection via thelumbar region or lumber area is also referred to as “lumbar IT delivery”or “lumbar IT administration.” The term “cisterna magna” refers to thespace around and below the cerebellum via the opening between the skulland the top of the spine. Typically, intrathecal injection via cisternamagna is also referred to as “cisterna magna delivery.” The term“cerebral ventricle” refers to the cavities in the brain that arecontinuous with the central canal of the spinal cord. Typically,injections via the cerebral ventricle cavities are referred to asintravetricular Cerebral (ICV) delivery.

In some embodiments, “intrathecal administration” or “intrathecaldelivery” according to the present invention refers to lumbar ITadministration or delivery, for example, delivered between the third andfourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1region of the spine. It is contemplated that lumbar IT administration ordelivery distinguishes over cisterna magna delivery in that lumbar ITadministration or delivery according to our invention provides betterand more effective delivery to the distal spinal canal, while cisternamagna delivery, among other things, typically does not deliver well tothe distal spinal canal.

Device for Intrathecal Delivery

Various devices may be used for intrathecal delivery according to thepresent invention. In some embodiments, a device for intrathecaladministration contains a fluid access port (e.g., injectable port); ahollow body (e.g., catheter) having a first flow orifice in fluidcommunication with the fluid access port and a second flow orificeconfigured for insertion into spinal cord; and a securing mechanism forsecuring the insertion of the hollow body in the spinal cord. As anon-limiting example shown in FIG. 62, a suitable securing mechanismcontains one or more nobs mounted on the surface of the hollow body anda sutured ring adjustable over the one or more nobs to prevent thehollow body (e.g., catheter) from slipping out of the spinal cord. Invarious embodiments, the fluid access port comprises a reservoir. Insome embodiments, the fluid access port comprises a mechanical pump(e.g., an infusion pump). In some embodiments, an implanted catheter isconnected to either a reservoir (e.g., for bolus delivery), or aninfusion pump. The fluid access port may be implanted or external

In some embodiments, intrathecal administration may be performed byeither lumbar puncture (i.e., slow bolus) or via a port-catheterdelivery system (i.e., infusion or bolus). In some embodiments, thecatheter is inserted between the laminae of the lumbar vertebrae and thetip is threaded up the thecal space to the desired level (generallyL3-L4) (FIG. 63).

Relative to intravenous administration, a single dose volume suitablefor intrathecal administration is typically small. Typically,intrathecal delivery according to the present invention maintains thebalance of the composition of the CSF as well as the intracranialpressure of the subject. In some embodiments, intrathecal delivery isperformed absent the corresponding removal of CSF from a subject. Insome embodiments, a suitable single dose volume may be e.g., less thanabout 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml, or 0.5ml. In some embodiments, a suitable single dose volume may be about0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 0.5-2 ml, 0.5-1 ml, 1-3 ml, 1-5 ml, 1.5-3ml, 1-4 ml, or 0.5-1.5 ml. In some embodiments, intrathecal deliveryaccording to the present invention involves a step of removing a desiredamount of CSF first. In some embodiments, less than about 10 ml (e.g.,less than about 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml) ofCSF is first removed before IT administration. In those cases, asuitable single dose volume may be e.g., more than about 3 ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

Various other devices may be used to effect intrathecal administrationof a therapeutic composition. For example, formulations containingdesired enzymes may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Lancet 2: 983-84, 1963). More specifically, in thismethod, a ventricular tube is inserted through a hole formed in theanterior horn and is connected to an Ommaya reservoir installed underthe scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions or formulations to anindividual are described in U.S. Pat. No. 6,217,552, incorporated hereinby reference. Alternatively, the drug may be intrathecally given, forexample, by a single injection, or continuous infusion. It should beunderstood that the dosage treatment may be in the form of a single doseadministration or multiple doses.

For injection, formulations of the invention can be formulated in liquidsolutions. In addition, the enzyme may be formulated in solid form andre-dissolved or suspended immediately prior to use. Lyophilized formsare also included. The injection can be, for example, in the form of abolus injection or continuous infusion (e.g., using infusion pumps) ofthe enzyme.

In one embodiment of the invention, the enzyme is administered bylateral cerebro ventricular injection into the brain of a subject. Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the enzyme and/or otherpharmaceutical formulation is administered through a surgically insertedshunt into the cerebral ventricle of a subject. For example, theinjection can be made into the lateral ventricles, which are larger. Insome embodiments, injection into the third and fourth smaller ventriclescan also be made.

In yet another embodiment, the pharmaceutical compositions used in thepresent invention are administered by injection into the cisterna magna,or lumbar area of a subject.

In another embodiment of the method of the invention, thepharmaceutically acceptable formulation provides sustained delivery,e.g., “slow release” of the enzyme or other pharmaceutical compositionused in the present invention, to a subject for at least one, two,three, four weeks or longer periods of time after the pharmaceuticallyacceptable formulation is administered to the subject.

As used herein, the term “sustained delivery” refers to continualdelivery of a pharmaceutical formulation of the invention in vivo over aperiod of time following administration, preferably at least severaldays, a week or several weeks. Sustained delivery of the composition canbe demonstrated by, for example, the continued therapeutic effect of theenzyme over time (e.g., sustained delivery of the enzyme can bedemonstrated by continued reduced amount of storage granules in thesubject). Alternatively, sustained delivery of the enzyme may bedemonstrated by detecting the presence of the enzyme in vivo over time.

Delivery to Target Tissues

As discussed above, one of the surprising and important features of thepresent invention is that therapeutic agents, in particular, replacementenzymes administered using inventive methods and compositions of thepresent invention are able to effectively and extensively diffuse acrossthe brain surface and penetrate various layers or regions of the brain,including deep brain regions. In addition, inventive methods andcompositions of the present invention effectively deliver therapeuticagents (e.g., an ASA enzyme) to various tissues, neurons or cells ofspinal cord, including the lumbar region, which is hard to target byexisting CNS delivery methods such as ICV injection. Furthermore,inventive methods and compositions of the present invention deliversufficient amount of therapeutic agents (e.g., an ASA enzyme) to bloodstream and various peripheral organs and tissues.

Thus, in some embodiments, a therapeutic protein (e.g., an ASA enzyme)is delivered to the central nervous system of a subject. In someembodiments, a therapeutic protein (e.g., an ASA enzyme) is delivered toone or more of target tissues of brain, spinal cord, and/or peripheralorgans. As used herein, the term “target tissues” refers to any tissuethat is affected by the lysosomal storage disease to be treated or anytissue in which the deficient lysosomal enzyme is normally expressed. Insome embodiments, target tissues include those tissues in which there isa detectable or abnormally high amount of enzyme substrate, for examplestored in the cellular lysosomes of the tissue, in patients sufferingfrom or susceptible to the lysosomal storage disease. In someembodiments, target tissues include those tissues that displaydisease-associated pathology, symptom, or feature. In some embodiments,target tissues include those tissues in which the deficient lysosomalenzyme is normally expressed at an elevated level. As used herein, atarget tissue may be a brain target tissue, a spinal cord target tissueand/or a peripheral target tissue. Exemplary target tissues aredescribed in detail below.

Brain Target Tissues

In general, the brain can be divided into different regions, layers andtissues. For example, meningeal tissue is a system of membranes whichenvelops the central nervous system, including the brain. The meningescontain three layers, including dura matter, arachnoid matter, and piamatter. In general, the primary function of the meninges and of thecerebrospinal fluid is to protect the central nervous system. In someembodiments, a therapeutic protein in accordance with the presentinvention is delivered to one or more layers of the meninges.

The brain has three primary subdivisions, including the cerebrum,cerebellum, and brain stem. The cerebral hemispheres, which are situatedabove most other brain structures and are covered with a cortical layer.Underneath the cerebrum lies the brainstem, which resembles a stalk onwhich the cerebrum is attached. At the rear of the brain, beneath thecerebrum and behind the brainstem, is the cerebellum.

The diencephalon, which is located near the midline of the brain andabove the mesencephalon, contains the thalamus, metathalamus,hypothalamus, epithalamus, prethalamus, and pretectum. Themesencephalon, also called the midbrain, contains the tectum,tegumentum, ventricular mesocoelia, and cerebral peduncels, the rednucleus, and the cranial nerve III nucleus. The mesencephalon isassociated with vision, hearing, motor control, sleep/wake, alertness,and temperature regulation.

Regions of tissues of the central nervous system, including the brain,can be characterized based on the depth of the tissues. For example, CNS(e.g., brain) tissues can be characterized as surface or shallowtissues, mid-depth tissues, and/or deep tissues.

According to the present invention, a therapeutic protein (e.g., areplacement enzyme) may be delivered to any appropriate brain targettissue(s) associated with a particular disease to be treated in asubject. In some embodiments, a therapeutic protein (e.g., a replacementenzyme) in accordance with the present invention is delivered to surfaceor shallow brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered tomid-depth brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered to deepbrain target tissue. In some embodiments, a therapeutic protein inaccordance with the present invention is delivered to a combination ofsurface or shallow brain target tissue, mid-depth brain target tissue,and/or deep brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered to a deepbrain tissue at least 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or morebelow (or internal to) the external surface of the brain.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more surface or shallow tissues of cerebrum. In some embodiments,the targeted surface or shallow tissues of the cerebrum are locatedwithin 4 mm from the surface of the cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are selected frompia matter tissues, cerebral cortical ribbon tissues, hippocampus,Virchow Robin space, blood vessels within the VR space, the hippocampus,portions of the hypothalamus on the inferior surface of the brain, theoptic nerves and tracts, the olfactory bulb and projections, andcombinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more deep tissues of the cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are located 4 mm(e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below (or internal to)the surface of the cerebrum. In some embodiments, targeted deep tissuesof the cerebrum include the cerebral cortical ribbon. In someembodiments, targeted deep tissues of the cerebrum include one or moreof the diencephalon (e.g., the hypothalamus, thalamus, prethalamus,subthalamus, etc.), metencephalon, lentiform nuclei, the basal ganglia,caudate, putamen, amygdala, globus pallidus, and combinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more tissues of the cerebellum. In certain embodiments, thetargeted one or more tissues of the cerebellum are selected from thegroup consisting of tissues of the molecular layer, tissues of thePurkinje cell layer, tissues of the Granular cell layer, cerebellarpeduncles, and combination thereof. In some embodiments, therapeuticagents (e.g., enzymes) are delivered to one or more deep tissues of thecerebellum including, but not limited to, tissues of the Purkinje celllayer, tissues of the Granular cell layer, deep cerebellar white mattertissue (e.g., deep relative to the Granular cell layer), and deepcerebellar nuclei tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more tissues of the brainstem. In some embodiments, the targetedone or more tissues of the brainstem include brain stem white mattertissue and/or brain stem nuclei tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered tovarious brain tissues including, but not limited to, gray matter, whitematter, periventricular areas, pia-arachnoid, meninges, neocortex,cerebellum, deep tissues in cerebral cortex, molecular layer,caudate/putamen region, midbrain, deep regions of the pons or medulla,and combinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered tovarious cells in the brain including, but not limited to, neurons, glialcells, perivascular cells and/or meningeal cells. In some embodiments, atherapeutic protein is delivered to oligodendrocytes of deep whitematter.

Spinal Cord

In general, regions or tissues of the spinal cord can be characterizedbased on the depth of the tissues. For example, spinal cord tissues canbe characterized as surface or shallow tissues, mid-depth tissues,and/or deep tissues.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more surface or shallow tissues of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cord islocated within 4 mm from the surface of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cordcontains pia matter and/or the tracts of white matter.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more deep tissues of the spinal cord. In some embodiments, atargeted deep tissue of the spinal cord is located internal to 4 mm fromthe surface of the spinal cord. In some embodiments, a targeted deeptissue of the spinal cord contains spinal cord grey matter and/orependymal cells.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toneurons of the spinal cord.

Peripheral Target Tissues

As used herein, peripheral organs or tissues refer to any organs ortissues that are not part of the central nervous system (CNS).Peripheral target tissues may include, but are not limited to, bloodsystem, liver, kidney, heart, endothelium, bone marrow and bone marrowderived cells, spleen, lung, lymph node, bone, cartilage, ovary andtestis. In some embodiments, a therapeutic protein (e.g., a replacementenzyme) in accordance with the present invention is delivered to one ormore of the peripheral target tissues.

Biodistribution and Bioavailability

In various embodiments, once delivered to the target tissue, atherapeutic agent (e.g., an ASA enzyme) is localized intracellularly.For example, a therapeutic agent (e.g., enzyme) may be localized toexons, axons, lysosomes, mitochondria or vacuoles of a target cell(e.g., neurons such as Purkinje cells). For example, in some embodimentsintrathecally-administered enzymes demonstrate translocation dynamicssuch that the enzyme moves within the perivascular space (e.g., bypulsation-assisted convective mechanisms). In addition, active axonaltransport mechanisms relating to the association of the administeredprotein or enzyme with neurofilaments may also contribute to orotherwise facilitate the distribution of intrathecally-administeredproteins or enzymes into the deeper tissues of the central nervoussystem.

In some embodiments, a therapeutic agent (e.g., an ASA enzyme) deliveredaccording to the present invention may achieve therapeutically orclinically effective levels or activities in various targets tissuesdescribed herein. As used herein, a therapeutically or clinicallyeffective level or activity is a level or activity sufficient to confera therapeutic effect in a target tissue. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). For example, atherapeutically or clinically effective level or activity may be anenzymatic level or activity that is sufficient to ameliorate symptomsassociated with the disease in the target tissue (e.g., GAG storage).

In some embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may achieve an enzymaticlevel or activity that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% of the normal level or activity of the correspondinglysosomal enzyme in the target tissue. In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may achieve an enzymatic level or activity that isincreased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold or 10-fold as compared to a control (e.g.,endogenous levels or activities without the treatment). In someembodiments, a therapeutic agent (e.g., a replacement enzyme) deliveredaccording to the present invention may achieve an increased enzymaticlevel or activity at least approximately 10 nmol/hr/mg, 20 nmol/hr/mg,40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg, 80nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600nmol/hr/mg in a target tissue.

In some embodiments, inventive methods according to the presentinvention are particularly useful for targeting the lumbar region. Insome embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may achieve an increasedenzymatic level or activity in the lumbar region of at leastapproximately 500 nmol/hr/mg, 600 nmol/hr/mg, 700 nmol/hr/mg, 800nmol/hr/mg, 900 nmol/hr/mg, 1000 nmol/hr/mg, 1500 nmol/hr/mg, 2000nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, or 10,000nmol/hr/mg.

In general, therapeutic agents (e.g., replacement enzymes) deliveredaccording to the present invention have sufficiently long half time inCSF and target tissues of the brain, spinal cord, and peripheral organs.In some embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may have a half-life of atleast approximately 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 12 hours, 16 hours, 18 hours, 20 hours, 25 hours, 30 hours, 35hours, 40 hours, up to 3 days, up to 7 days, up to 14 days, up to 21days or up to a month. In some embodiments, In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may retain detectable level or activity in CSF orbloodstream after 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90hours, 96 hours, 102 hours, or a week following administration.Detectable level or activity may be determined using various methodsknown in the art.

In certain embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention achieves a concentration ofat least 30 μg/ml in the CNS tissues and cells of the subject followingadministration (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30minutes, or less, following intrathecal administration of thepharmaceutical composition to the subject). In certain embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention achieves a concentration of at least 20 μg/ml, atleast 15 μg/ml, at least 10 μg/ml, at least 7.5 μg/ml, at least 5 μg/ml,at least 2.5 μg/ml, at least 1.0 μg/ml or at least 0.5 μg/ml in thetargeted tissues or cells of the subject (e.g., brain tissues orneurons) following administration to such subject (e.g., one week, 3days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less followingintrathecal administration of such pharmaceutical compositions to thesubject).

Treatment of Metachromatic Leukodystrophy Disease (MLD)

Metachromatic Leukodystrophy Disease (MLD), is an autosomal recessivedisorder resulting from a deficiency of the enzyme Arylsulfatease A(ASA). ASA, which is encoded by the ARSA gene in humans, is an enzymethat breaks down cerebroside 3-sulfate or sphingolipid3-O-sulfogalactosylceramide (sulfatide) into cerebroside and sulfate. Inthe absence of the enzyme, sulfatides accumulate in the nervous system(e.g., myelin sheaths, neurons and glial cells) and to a lesser extentin visceral organs. The consequence of these molecular and cellularevents is progressive demyelination and axonal loss within the CNS andPNS, which is accompanied clinically by severe motor and cognitivedysfunction.

A defining clinical feature of this disorder is central nervous system(CNS) degeneration, which results in cognitive impairment (e.g., mentalretardation, nervous disorders, and blindness, among others).

MLD can manifest itself in young children (Late-infantile form), whereaffected children typically begin showing symptoms just after the firstyear of life (e.g., at about 15-24 months), and generally do not survivepast the age of 5 years. MLD can manifest itself in children (Juvenileform), where affected children typically show cognitive impairment byabout the age of 3-10 years, and life-span can vary (e.g., in the rangeof 10-15 years after onset of symptoms). MLD can manifest itself inadults (Adult-onset form) and can appear in individuals of any age(e.g., typically at age 16 and later) and the progression of the diseasecan vary greatly.

Compositions and methods of the present invention may be used toeffectively treat individuals suffering from or susceptible to MLD. Theterms, “treat” or “treatment,” as used herein, refers to amelioration ofone or more symptoms associated with the disease, prevention or delay ofthe onset of one or more symptoms of the disease, and/or lessening ofthe severity or frequency of one or more symptoms of the disease.Exemplary symptoms include, but are not limited to, intracranialpressure, hydrocephalus ex vacuo, accumulated sulfated glycolipids inthe myelin sheaths in the central and peripheral nervous system and invisceral organs, progressive demyelination and axonal loss within theCNS and PNS, and/or motor and cognitive dysfunction.

In some embodiments, treatment refers to partially or completealleviation, amelioration, relief, inhibition, delaying onset, reducingseverity and/or incidence of neurological impairment in an MLD patient.As used herein, the term “neurological impairment” includes varioussymptoms associated with impairment of the central nervous system (e.g.,the brain and spinal cord). In some embodiments, various symptoms of MLDare associated with impairment of the peripheral nervous system (PNS).In some embodiments, neurological impairment in an MLD patient ischaracterized by decline in gross motor function. It will be appreciatedthat gross motor function may be assessed by any appropriate method. Forexample, in some embodiments, gross motor function is measured as thechange from a baseline in motor function using the Gross Motor FunctionMeasure-88 (GMFM-88) total raw score.

In some embodiments, treatment refers to decreased sulfatideaccumulation in various tissues. In some embodiments, treatment refersto decreased sulfatide accumulation in brain target tissues, spinal cordneurons, and/or peripheral target tissues. In certain embodiments,sulfatide accumulation is decreased by about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% or more as compared to a control. In some embodiments, sulfatideaccumulation is decreased by at least 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to acontrol. It will be appreciated that sulfatide storage may be assessedby any appropriate method. For example, in some embodiments, sulfatidestorage is measured by alcian blue staining In some embodiments,sulfatide storage is measured by LAMP-1 staining.

In some embodiments, treatment refers to reduced vacuolization inneurons (e.g., neurons containing Purkinje cells). In certainembodiments, vacuolization in neurons is decreased by about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100% or more as compared to a control. In someembodiments, vacuolization is decreased by at least 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold ascompared to a control.

In some embodiments, treatment refers to increased ASA enzyme activityin various tissues. In some embodiments, treatment refers to increasedASA enzyme activity in brain target tissues, spinal cord neurons and/orperipheral target tissues. In some embodiments, ASA enzyme activity isincreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900% 1000% or more as compared to a control. In someembodiments, ASA enzyme activity is increased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or10-fold as compared to a control. In some embodiments, increased ASAenzymatic activity is at least approximately 10 nmol/hr/mg, 20nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg,80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg, 600nmol/hr/mg or more. In some embodiments, ASA enzymatic activity isincreased in the lumbar region. In some embodiments, increased ASAenzymatic activity in the lumbar region is at least approximately 2000nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, 10,000nmol/hr/mg, or more.

In some embodiments, treatment refers to decreased progression of lossof cognitive ability. In certain embodiments, progression of loss ofcognitive ability is decreased by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% ormore as compared to a control. In some embodiments, treatment refers todecreased developmental delay. In certain embodiments, developmentaldelay is decreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more ascompared to a control.

In some embodiments, treatment refers to increased survival (e.g.survival time). For example, treatment can result in an increased lifeexpectancy of a patient. In some embodiments, treatment according to thepresent invention results in an increased life expectancy of a patientby more than about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 100%, about 105%, about 110%, about 115%, about 120%, about125%, about 130%, about 135%, about 140%, about 145%, about 150%, about155%, about 160%, about 165%, about 170%, about 175%, about 180%, about185%, about 190%, about 195%, about 200% or more, as compared to theaverage life expectancy of one or more control individuals with similardisease without treatment. In some embodiments, treatment according tothe present invention results in an increased life expectancy of apatient by more than about 6 month, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, about2 years, about 3 years, about 4 years, about 5 years, about 6 years,about 7 years, about 8 years, about 9 years, about 10 years or more, ascompared to the average life expectancy of one or more controlindividuals with similar disease without treatment. In some embodiments,treatment according to the present invention results in long termsurvival of a patient. As used herein, the term “long term survival”refers to a survival time or life expectancy longer than about 40 years,45 years, 50 years, 55 years, 60 years, or longer.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with the same form MLD (e.g., late-infantile,juvenile, or adult-onset form), who is about the same age and/or genderas the individual being treated (to ensure that the stages of thedisease in the treated individual and the control individual(s) arecomparable).

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having MLD or having the potential to develop MLD. The individualcan have residual endogenous ASA expression and/or activity, or nomeasurable activity. For example, the individual having MLD may have ASAexpression levels that are less than about 30-50%, less than about25-30%, less than about 20-25%, less than about 15-20%, less than about10-15%, less than about 5-10%, less than about 0.1-5% of normal ASAexpression levels.

In some embodiments, the individual is an individual who has beenrecently diagnosed with the disease. Typically, early treatment(treatment commencing as soon as possible after diagnosis) is importantto minimize the effects of the disease and to maximize the benefits oftreatment.

Immune Tolerance

Generally, intrathecal administration of a therapeutic agent (e.g., areplacement enzyme) according to the present invention does not resultin severe adverse effects in the subject. As used herein, severe adverseeffects induce, but are not limited to, substantial immune response,toxicity, or death. As used herein, the term “substantial immuneresponse” refers to severe or serious immune responses, such as adaptiveT-cell immune responses.

Thus, in many embodiments, inventive methods according to the presentinvention do not involve concurrent immunosuppressant therapy (i.e., anyimmunosuppressant therapy used as pre-treatment/pre-conditioning or inparallel to the method). In some embodiments, inventive methodsaccording to the present invention do not involve an immune toleranceinduction in the subject being treated. In some embodiments, inventivemethods according to the present invention do not involve apre-treatment or preconditioning of the subject using T-cellimmunosuppressive agent.

In some embodiments, intrathecal administration of therapeutic agentscan mount an immune response against these agents. Thus, in someembodiments, it may be useful to render the subject receiving thereplacement enzyme tolerant to the enzyme replacement therapy. Immunetolerance may be induced using various methods known in the art. Forexample, an initial 30-60 day regimen of a T-cell immunosuppressiveagent such as cyclosporin A (CsA) and an antiproliferative agent, suchas, azathioprine (Aza), combined with weekly intrathecal infusions oflow doses of a desired replacement enzyme may be used.

Any immunosuppressant agent known to the skilled artisan may be employedtogether with a combination therapy of the invention. Suchimmunosuppressant agents include but are not limited to cyclosporine,FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such as etanercept (seee.g. Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; Nevins,2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J.Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226;Potter et al., 1999, Ann. N.Y. Acad. Sci. 875, 159-174; Slavik et al.,1999, Immunol. Res. 19, 1-24; Gaziev et al., 1999, Bone MarrowTransplant. 25, 689-696; Henry, 1999, Clin. Transplant. 13, 209-220;Gummert et al., 1999, J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al.,2000, Transplantation 69, 1275-1283). The anti-IL2 receptor(.alpha.-subunit) antibody daclizumab (e.g. Zenapax™), which has beendemonstrated effective in transplant patients, can also be used as animmunosuppressant agent (see e.g. Wiseman et al., 1999, Drugs 58,1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619;Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard et al., 1999,Pharmacotherapy 19, 1127-1137; Eckhoff et al., 2000, Transplantation 69,1867-1872; Ekberg et al., 2000, Transpl. Int. 13, 151-159).Additionalimmunosuppressant agents include but are not limited toanti-CD2 (Branco et al., 1999, Transplantation 68, 1588-1596; Przepiorkaet al., 1998, Blood 92, 4066-4071), anti-CD4 (Marinova-Mutafchieva etal., 2000, Arthritis Rheum. 43, 638-644; Fishwild et al., 1999, Clin.Immunol. 92, 138-152), and anti-CD40 ligand (Hong et al., 2000, Semin.Nephrol. 20, 108-125; Chirmule et al., 2000, J. Virol. 74, 3345-3352;Ito et al., 2000, J. Immunol. 164, 1230-1235).

Administration

Inventive methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of thetherapeutic agents (e.g., replacement enzymes) described herein.Therapeutic agents (e.g., replacement enzymes) can be administered atregular intervals, depending on the nature, severity and extent of thesubject's condition (e.g., a lysosomal storage disease). In someembodiments, a therapeutically effective amount of the therapeuticagents (e.g., replacement enzymes) of the present invention may beadministered intrathecally periodically at regular intervals (e.g., onceevery year, once every six months, once every five months, once everythree months, bimonthly (once every two months), monthly (once everymonth), biweekly (once every two weeks), weekly).

In some embodiments, intrathecal administration may be used inconjunction with other routes of administration (e.g., intravenous,subcutaneously, intramuscularly, parenterally, transdermally, ortransmucosally (e.g., orally or nasally)). In some embodiments, thoseother routes of administration (e.g., intravenous administration) may beperformed no more frequent than biweekly, monthly, once every twomonths, once every three months, once every four months, once every fivemonths, once every six months, annually administration.

As used herein, the term “therapeutically effective amount” is largelydetermined base on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). For example, atherapeutically effective amount may be an amount sufficient to achievea desired therapeutic and/or prophylactic effect, such as an amountsufficient to modulate lysosomal enzyme receptors or their activity tothereby treat such lysosomal storage disease or the symptoms thereof(e.g., a reduction in or elimination of the presence or incidence of“zebra bodies” or cellular vacuolization following the administration ofthe compositions of the present invention to a subject). Generally, theamount of a therapeutic agent (e.g., a recombinant lysosomal enzyme)administered to a subject in need thereof will depend upon thecharacteristics of the subject. Such characteristics include thecondition, disease severity, general health, age, sex and body weight ofthe subject. One of ordinary skill in the art will be readily able todetermine appropriate dosages depending on these and other relatedfactors. In addition, both objective and subjective assays mayoptionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration of the treatment; andlike factors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg brain weight to 500 mg/kg brain weight, e.g., fromabout 0.005 mg/kg brain weight to 400 mg/kg brain weight, from about0.005 mg/kg brain weight to 300 mg/kg brain weight, from about 0.005mg/kg brain weight to 200 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 100 mg/kg brain weight, from about 0.005 mg/kg brainweight to 90 mg/kg brain weight, from about 0.005 mg/kg brain weight to80 mg/kg brain weight, from about 0.005 mg/kg brain weight to 70 mg/kgbrain weight, from about 0.005 mg/kg brain weight to 60 mg/kg brainweight, from about 0.005 mg/kg brain weight to 50 mg/kg brain weight,from about 0.005 mg/kg brain weight to 40 mg/kg brain weight, from about0.005 mg/kg brain weight to 30 mg/kg brain weight, from about 0.005mg/kg brain weight to 25 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 20 mg/kg brain weight, from about 0.005 mg/kg brainweight to 15 mg/kg brain weight, from about 0.005 mg/kg brain weight to10 mg/kg brain weight.

In some embodiments, the therapeutically effective dose is greater thanabout 0.1 mg/kg brain weight, greater than about 0.5 mg/kg brain weight,greater than about 1.0 mg/kg brain weight, greater than about 3 mg/kgbrain weight, greater than about 5 mg/kg brain weight, greater thanabout 10 mg/kg brain weight, greater than about 15 mg/kg brain weight,greater than about 20 mg/kg brain weight, greater than about 30 mg/kgbrain weight, greater than about 40 mg/kg brain weight, greater thanabout 50 mg/kg brain weight, greater than about 60 mg/kg brain weight,greater than about 70 mg/kg brain weight, greater than about 80 mg/kgbrain weight, greater than about 90 mg/kg brain weight, greater thanabout 100 mg/kg brain weight, greater than about 150 mg/kg brain weight,greater than about 200 mg/kg brain weight, greater than about 250 mg/kgbrain weight, greater than about 300 mg/kg brain weight, greater thanabout 350 mg/kg brain weight, greater than about 400 mg/kg brain weight,greater than about 450 mg/kg brain weight, greater than about 500 mg/kgbrain weight.

In some embodiments, the therapeutically effective dose may also bedefined by mg/kg body weight. As one skilled in the art wouldappreciate, the brain weights and body weights can be correlated.Dekaban A S. “Changes in brain weights during the span of human life:relation of brain weights to body heights and body weights,” Ann Neurol1978; 4:345-56. Thus, in some embodiments, the dosages can be convertedas shown in Table 5.

TABLE 5 Correlation between Brain Weights, body weights and ages ofmales Age (year) Brain weight (kg) Body weight (kg) 3 (31-43 months)1.27 15.55 4-5 1.30 19.46

In some embodiments, the therapeutically effective dose may also bedefined by mg/15 cc of CSF. As one skilled in the art would appreciate,therapeutically effective doses based on brain weights and body weightscan be converted to mg/15 cc of CSF. For example, the volume of CSF inadult humans is approximately 150 mL (Johanson C E, et al. “Multiplicityof cerebrospinal fluid functions: New challenges in health and disease,”Cerebrospinal Fluid Res. 2008 May 14; 5:10). Therefore, single doseinjections of 0.1 mg to 50 mg protein to adults would be approximately0.01 mg/15 cc of CSF (0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses inadults.

It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the enzyme replacement therapy andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed invention.

Kits

The present invention further provides kits or other articles ofmanufacture which contains the formulation of the present invention andprovides instructions for its reconstitution (if lyophilized) and/oruse. Kits or other articles of manufacture may include a container, anIDDD, a catheter and any other articles, devices or equipment useful ininterthecal administration and associated surgery. Suitable containersinclude, for example, bottles, vials, syringes (e.g., pre-filledsyringes), ampules, cartridges, reservoirs, or lyo-jects. The containermay be formed from a variety of materials such as glass or plastic. Insome embodiments, a container is a pre-filled syringe. Suitablepre-filled syringes include, but are not limited to, borosilicate glasssyringes with baked silicone coating, borosilicate glass syringes withsprayed silicone, or plastic resin syringes without silicone.

Typically, the container may holds formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to protein concentrations as describedabove. The label may further indicate that the formulation is useful orintended for, for example, IT administration. In some embodiments, acontainer may contain a single dose of a stable formulation containing atherapeutic agent (e.g., a replacement enzyme). In various embodiments,a single dose of the stable formulation is present in a volume of lessthan about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml,1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a container holding theformulation may be a multi-use vial, which allows for repeatadministrations (e.g., from 2-6 administrations) of the formulation.Kits or other articles of manufacture may further include a secondcontainer comprising a suitable diluent (e.g., BWFI, saline, bufferedsaline). Upon mixing of the diluent and the formulation, the finalprotein concentration in the reconstituted formulation will generally beat least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 25mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml). Kitsor other articles of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, IDDDs, catheters, syringes, andpackage inserts with instructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLES Example 1 Toxicology of IT Administered Arylsulfatase A

To assess the ability of other intrathecally-administered recombinantenzymes to distribute into the cells and tissues of the CNS, GLP studywas conducted to evaluate repeat dose intrathecal (IT) administration ofrecombinantly-prepared human arylsulfatase A (rhASA) from a toxicologyand safety pharmacology perspective over a one-month period in juvenile(less than 12 months of age) cynomolgus monkeys. The formulation ofrhASA was prepared and formulated in a vehicle of 154 mM NaCl, 0.005%polysorbate 20 at a pH of 6.0.

To achieve this, nine male and nine female juvenile cynomolgus monkeyswere randomly assigned by body weight to one of three treatment groupsas shown in the following Table 6. The animals (with the exception of 1male animal for Dose 1) received 0.6 mL short-term IT infusion of 0, 3or 31 mg/mL of rhASA (total dose of 0, 1.8 or 18.6 mg) every other weekfor a total of three doses per animal. Body weights, clinicalobservations, neurological and physical examinations, clinicalpathology, ophthalmologic examinations, and toxicokinetic sampling weremonitored. All of the animals were necropsied on Day 29, 30 or 31 (−24hours after the last IT dose). Selected tissues were harvested, savedand examined microscopically.

TABLE 6 Nominal Dose Dose Administered Number of Concentration VolumeDose Group Animals (mg/mL) (mL) (mg) 1 3M, 3F 0 0.6 0 2 3M, 3F 3 0.6 1.83 3M, 3F 31 0.6 18.6

The concentrations of rhASA detected in the CNS tissues of thecynomolgus monkeys were analyzed by ELISA and compared to a therapeutictarget of 10% of normal human rhASA concentrations, corresponding toapproximately 2.5 ng/mg of tissue. Tissue samples or punches wereextracted from different areas of the brains of the cynomolgus monkeysand further analyzed for the presence of rhASA. FIG. 24 illustrates thetissues from which the punches were extracted. The punched tissuesamples reflected an increase in the concentrations of rhASA, asreflected in FIGS. 25A-G, with a deposition gradient from the cerebralcortex to the deep white matter and deep gray matter.

Concentrations of rhASA detected using the same punch from both the ITand ICV routes of administration for six monkeys administered the 18.6mg dose of rhASA, are illustrated in FIGS. 26A-B. The concentrations ofrhASA detected in the deep white matter (FIG. 25A) and in the deep greymatter (FIG. 26B) brain tissues of adult and juvenile cynomolgus monkeysintrathecally—(IT) or intracerebroventricularly—(ICV) administered rhASAwere comparable.

The punched tissue samples extracted from the brains of adult andjuvenile cynomolgus monkeys were then analyzed to determine theconcentrations of rhASA deposited in the extracted tissue sample, and tocompare such concentrations to the therapeutic target concentration of2.5 ng rhASA per mg protein (corresponding to 10% of the normalconcentration of rhASA in a healthy subject). As illustrated in FIG.27A, in each tissue sample punch analyzed the 18.6 mg dose ofIT-administered rhASA resulted in an rhASA concentration which exceededthe target therapeutic concentration of 2.5 ng/mg of protein. Similarly,when a 1.8 mg dose of rhASA was IT-administered to juvenile cynomolgusmonkeys, each tissue sample punch analyzed demonstrated a concentrationof rhASA either within or exceeding the therapeutic concentration of 2.5ng/mg of protein and the median rhASA concentrations were above thetherapeutic target for all tissue punches tested (FIG. 27B).

To determine whether IT-administered rhASA was distributing to therelevant cells, tissue was analyzed from the deep white matter of acynomolgus monkey IT-administered 1.8 mg of ASA, from the areaillustrated in FIG. 28A. Immunostaining of the deep white matter tissuerevealed distribution of rhASA in the cynomolgus monkey inoligodendrocyte cells, as illustrated by FIG. 28B. Similarly, FIG. 28Cillustrates that the IT-administered rhrASA demonstrated co-localizationin the deep white matter tissues of the cynomolgus monkey. Inparticular, under staining co-localization in target organelles, such asthe lysosome, is evident (FIG. 28C), supporting the conclusion thatIT-administered rhASA is capable of distributing to the relevant cells,tissues and organelles of the CNS, including the lysosomes ofoligodendrocytes. The foregoing supports the conclusion that thedifference between ICV and IT delivery was also found to be minimal forrhASA delivery.

Example 2 Biodistribution with Radio-Labeled Protein

rhASA labeled with the positron emitter ¹²⁴I was prepared and formulatedin a vehicle of 154 mM NaCl, 0.005% polysorbate 20 at a pH of 6.0. Avolume of the formulation equivalent to 3 mg of rhASA (corresponding toapproximately 38 mg/kg of brain) was administered to adult cynomolgusmonkeys via intracerebroventricular (ICV) and intrathecal (IT) routes ofadministration. The cynomolgus monkeys were subject to high-resolutionPET scan imaging studies (microPET P4) to determine distribution of theadministered ¹²⁴I-labeled rhASA.

PET imaging data (FIG. 29) illustrates that both the ICV- andIT-administered ¹²⁴I-labeled rhASA effectively distributed to thetissues of the CNS, and in particular the ¹²⁴I-labeled rhASAadministered through the IT-lumbar catheter immediately and uniformlyspread in the cerebrospinal fluid (CSF) over the length of the spine. Inparticular, as depicted in FIG. 29, following ICV- andIT-administration, therapeutic concentrations of ¹²⁴I-labeled rhASA weredetected in the CNS tissues of the subject cynomolgus monkey, includingthe brain, spinal cord and CSF. The concentrations of rhASA detected insuch CNS tissues, and in particular in the tissues of the brain,exceeded the therapeutic target concentration of 2.5 ng/mg of protein.

While the distribution of rhASA protein was comparable for both IT andICV routes of administration, ICV resulted in notably less depositionwithin the spinal column, as evidence by FIG. 29.

Twenty four hours following administration of the formulation, both theICV- and IT-administered ¹²⁴I-labeled ASA effectively distributed to thetissues of the CNS. In particular, twenty four hours followingIT-administration 12.4% of the administered dose was in the cranialregion, compared to 16.7% of the ICV-administered dose. Accordingly, theconcentrations of rhASA detected in such CNS tissues, and in particularin the tissues of the brain, when rhASA was administered IT approachedthose concentrations detected following ICV-administration of the samedose.

ICV injection of the ¹²⁴I-labeled rhASA results ICV injection results inthe immediate transfer of the injected volume to the cisterna magna,cisterna pontis, cisterna interpeduncularis and proximal spine, asillustrated in FIG. 30. As also illustrated in FIG. 30, within 2-5 hr ITadministration delivered the ¹²⁴I-labeled rhASA to the same initialcompartments (cisternae and proximal spine) as shown for the ICVadministration. Twenty four hours following both ICV- andIT-administration distribution of the ¹²⁴I-labeled rhASA was comparable,as illustrated in FIG. 31. Accordingly, unlike small molecules drugs,the foregoing results suggest that ICV-administration offers minimaladvantages over IT-administration of rhASA.

These results confirm that rhASA can be delivered to a subject using theless invasive IT route of administration and thereby achieve therapeuticconcentrations in target cells and tissues.

The lysosomal storage diseases represent a family of genetic disorderscaused by missing or defective enzymes which result in abnormalsubstrate accumulation. While the peripheral symptoms associated withseveral of these diseases can be effectively mitigated by intravenousadministration of recombinant enzymes, intravenous administration ofsuch recombinant enzymes are not expected to significantly impact theCNS manifestations associated with a majority of the lysosomal storagedisease. For example, recombinant human iduronate-2-sulfatase(Idursulfase, Elaprase®; Shire Human Genetic Therapies, Inc. Lexington,Mass.) is approved for treatment of the somatic symptoms of Huntersyndrome but there is no pharmacologic therapy for the treatment of theneurologic manifestations which can include delayed development andprogressive mental impairment. This is in part due to the nature of I2S,which is a large, highly-glycosylated enzyme with a molecular weight ofapproximately 76 kD and that does not traverse the blood brain barrierfollowing intravenous administration.

The present inventors have therefore undertaken a program to investigatethe intrathecal (IT) delivery of intrathecal formulations of recombinanthuman enzymes, such as, for example, iduronate-2-sulfatase (I2S),arylsulfatase A (rhASA) and alpha-N-acetylglucosaminidase (Naglu). Theresults presented herein represent the first to demonstrate thatIT-lumbar administration of a recombinant lysosomal proteins result inthe delivery of a significant fraction of the administered protein tothe brain and in particular result in the widespread deposition of suchproteins in neurons of the brain and spinal cord in both cynomolgusmonkeys and dogs. Immunohistochemical analyses of the CNS tissuesdemonstrated that the protein is targeted to the lysosome, the site ofpathologic glycosaminoglycan accumulation in the lysosomal storagedisorders. Furthermore, the morphologic improvements demonstrated in theIKO mouse model of Hunter syndrome, the Naglu-deficient mouse model ofSanfilippo syndrome type B, and the ASA knockout mouse model ofmetachromatic leukodystrohpy (MLD) reinforces the observation thatIT-administered enzyme is distributed to the appropriate tissues andtransported to the appropriate cellular compartments and organelles.

The similarities observed in brain distribution patterns detected afterIT-lumbar and ICV administration of I2S is suggestive of bulk flow andactive remixing of the CSF. Thus in a clinical setting, both the IT andthe ICV administration routes are potentially feasible, however, theobserved deposition of I2S in the spinal cord following ITadministration provides a clear advantage in addressing spinal sequelaeand components of lysosomal storage diseases such as Hunter syndrome.Moreover, spinal injection ports are less invasive and expected to bemore suitable for chronic use, especially in pediatric subjects.

Evidence from perivascular cell staining and protein translocationdynamics observed by the foregoing PET imaging studies indicate thatenzyme moves within the perivascular space, presumably bypulsation-assisted convective mechanisms. An additional mechanism oftransport is suggested by the observed association of I2S withneurofilaments, indicative of active axonal transport. The latterpresumably begins with protein interaction with neuronalmannose-6-phosphate (M6P) receptors, which are widely expressed on cellsof the spinal cord and brain and which upon direct administration to thebrain parenchyma may cause I2S enzyme to be readily absorbed by targetcells. (Begley, et al., Curr Pharm Des (2008) 14: 1566-1580).

While axonal transport of lysosomal enzymes have previously been impliedby indirect methods in vivo and by imaging in vitro, the current studiesprovide the first direct evidence of axonal transport of non-virally orexpressed enzymes delivered via the CSF. Thus, protein delivery from theCSF to the brain surface and deeper into the brain tissues seems todepend on active transfer processes, none of which have been previouslydescribed or elucidate for protein or enzyme delivery to the cells,tissues and organelles of the brain.

Contrary to the prevailing viewpoint that the flow dynamics of theparenchyma interstitium and CSF would prevent the distribution ofIT-lumbar administered proteins to the white matter of the brain, theinstant studies clearly demonstrate that IT delivery of a lysosomalenzyme results in protein distribution and accumulation in all braintissues and deposition in the lysosomal compartment of target cellswhich are the site of pathologic glycosaminoglycan accumulation. (See,e.g., Fenstermacher et al., Ann N Y Acad Sci (1988) 531:29-39 andDiChiro et al., Neurology (1976) 26:1-8.) Together with the lessinvasive nature of IT-lumbar delivery, this route offers a clinicallyrelevant means of delivering biologic therapeutics to the brain,particularly in children.

Example 3 Formulations of Arylsulfatase A for It Administration

This example summarizes the work to establish a high concentrationliquid dosage form of rhASA (arylsulfase A) and the formulation of drugsubstance and drug product for treatment of Metachromatoc Leukodystrophy(MLD) via the intrathecal (IT) route of administration.

The stability data demonstrate that the saline formulation of drugsubstance and drug product (without PBS 20) is stable after 18 months at<−65 degrees C. and 18 months at 2-8 degrees C. During thepharmaceutical development of this protein, the solubility and stabilityof rhASA was investigated under limited buffer and excipient conditionsdue to its intended delivery to the CNS. Previously, formulationdevelopment studies had been conducted to develop an intravenous (IV)formulation. Based on the results of these experiments, a formulationcontaining 30 mg/ml of rhASA in 10 mM citrate-phosphate buffer, pH 5.5with 137 mM NaCl and 0.15% poloxomer 188 was selected as the lead IVformulation. rhASA was also formulated for IT delivery in threeformulations and stability data for this protein was investigated underthese conditions. rhASA lots derived from upstream material product atone site were utilized. The results demonstrated that rhASA was stablein 154 mM sodium chloride solution with 0.005% polysorbate 20 (P20), pH6.0 for at least 18 months at 2-8 degrees C. In addition, studies havebeen performed to demonstrate stability toward freeze-thaw andagitation-induced degradation.

Development lots were purified, ultrafiltered and diafiltered (UF/DF)into 10 mM citrate/phosphate, 137 mM NaCl, pH 5.5 with subsequent UF/DFinto final saline solution at a concentration of approximately 40 mg/mL.The UF/DF operations are summarized in Table 7.

TABLE 7 Selected Formulations for UF/DF Operations fromXcellerex-Derived Formu- lation Initial Buffer and UF/DF into SalineAdditive A 10 mM citrate/phosphate, 137 mM NaCl, pH 5.5. 0.005%Subsequent UF/DF into 154 mM NaCl. polysorbate Final pH 5.9 20* B 10 mMcitrate/phosphate, 137 mM NaCl, pH 5.5. 0.005% Subsequent UF/DF into 5mM sodium phosphate, polysorbate 145 mM NaCl, pH 6.0. Final pH 6.0 20* C10 mM citrate/phosphate, 137 mM NaCl, pH 5.5. 0.005% Subsequent UF/DFinto 10 mM citrate/phosphate, polysorbate 137 mM NaCl, pH 7.0, and asecond UF/DF into 20* 154 mM NaCl. Final pH 6.5rhASA

rhASA formulated at 40 mg/mL rhASA in 10 mM citrate sodium phosphatewith 137 mM NaCl, at pH 5.6 was dialyzed into five formulations whichwere utilized for IT preformulation studies (Table 8).

TABLE 8 Selected Buffers for IT Compatible Formulation ScreeningFormulation Number Buffer Species pH 1 154 mM NaCl * 5.9 2 154 mM NaCl** 7.0 3  5 mM phosphate buffer with 145 mM NaCl 6.0 4  5 mM phosphatebuffer with 145 mM NaCl 7.0 5  1 mM phosphate buffer with 2 mM CaCl₂ and7.0 137 mM NaCl

Methods

For melting temperature (Tm) determination by Differential Scanningcalorimetry (DSC), a capillary DSC microcalorimeter (MicroCal) wasemployed at a scan rate of 60° C./hr and a temperature range of 10-110°C. Buffer baselines were subtracted from the protein scans. The scanswere normalized for the protein concentration of each sample (measuredby ultraviolet absorbance at 280 nm and using an extinction coefficientof 0.69 (mg/mL)-1.cm⁻¹). For initial short-term stability experiments,rhASA drug substance was subjected to either two weeks at 40° C. or onemonth at 40° C. Additional samples were placed on short term stabilityat 2-8° C. for 3 months. Samples were filtered (Millipore, P/NSLGV033RS) and aliquots of 0.5 mL were dispensed into 2 mL with 13 mmFluorotec stoppers.

The effect of formulation composition (Table 8) on the Tm (temperaturemidpoint of the thermally induces denaturation) was investigated usingDSC. The Tm values for different formulation compositions are shown inFIG. 5. The Tm values exhibited similar unfolding temperatures for mostof the formulations, except low Tm values were observed for rhASAformulated in either 5 mM sodium phosphate with 154 mM NaCl at pH 7.0 or1 mM sodium phosphate with 2 mM CaCl₂ and 137 mM NaCl at pH 7.0.

The effect of thermal induced degradation of rhASA in the five selectedformulations (Table 8) was also investigated. Samples were stored eitherfor 2 weeks or one month at 40° C. or for 3 months at 2-8° C. SDS-PAGE(Coomassie) analysis of samples stored for 2 weeks at 40° C. detectedfragmentation of rhASA formulated in 5 mM sodium phosphate with 154 mMNaCl at pH 7.0 as well as in 1 mM sodium phosphate with 2 mM CaCl₂ and137 mM NaCl at pH 7.0 (FIG. 6). No such degradation was observed for theother formulations.

The presence of breakdown products is consistent with the lower percentmain peak observed by RP-HPLC for the same time points (Table 10). Itwas also observed that rhASA formulated in 1 mM PBS with 2 mM CaCl₂ atpH 7.0 did not maintain its pH at the onset and following the short termexposure to thermal stress conditions.

Waters HPLC systems were used for size exclusion and reversed phase HPLCanalyses. For initial SEC-HPLC analysis, 50 μg of rhASA was injected onto an Agilent Zorbax GF-250 column (4.6 mm×250 mm) and run isocraticallyat 0.24 mL/min using a mobile phase of 100 mM sodium citrate pH 5.5(octomer detection) with a detection wavelength of 280 nm. The analyseswere repeated using mobile phase conditions of 100 mM sodium citrate, pH7.0 (dimer detection).

All buffer exchange and concentration studies were performed usingCentricon-Plus 20 (Millipore, 10 kDa MWCO).

Preformulation Screening Studies—Effect of Buffer Species and pH

Due to the limited number of approved solution compositions used for CNSadministration, only five isotonic solution compositions, as listed inTable 8, were selected for screening.

pH Memory

Prior to the selection of buffers for long term stability, two “pHmemory” experiments were performed to investigate if the proteinbuffer-exchanged into saline solution was capable of maintaining the pHof the original buffer. In the initial experiment, rhASA atapproximately 8 mg/mL, was first dialyzed into 10 mM citrate-phosphatewith 137 mM NaCl, at either a pH value of 5.5 or 7.0, followed by asecond dialysis into saline solution. In the second experiment, rhASAwas dialyzed into 10 mM citrate-phosphate with 137 mM NaCl, at either pHvalues of 5.5 or 7.0 and subsequently buffer exchanged and concentratedinto saline solutions to approximately 35 mg/mL.

When rhASA formulated in 10 mM citrate-phosphate with 137 mM NaCl ateither pH values of 5.5 or 7.0 was dialyzed into saline solution, noincreased turbidity was observed. The pH of the final saline solutionwas similar to the pH of the previous citrate-phosphate buffer to whichit was exposed. When rhASA formulated in citrate-phosphate based buffersat either pH values of 5.5 or pH 7.0 were dialyzed into saline and thenconcentrated to approximately 35 mg/mL using a Centricon, the pH of theprotein saline solutions shifted from pH 5.5 to 5.8 or from pH 7.0 to6.8, respectively. Both concentrated rhASA solutions in saline wereslightly opalescent and had OD320 values in the range of 0.064 (pH 6.8)to 0.080 (pH 5.5).

Excipient Selection

Polysorbate 20 (P20) was included in all five selected solutioncompositions at a final concentration of 0.005%. The surfactant choicewas made based on prior experience of the in vivo tolerability of P20 at0.005% for CNS delivery of other Shire proteins. A solution of 5% P20(v/v) was prepared and the appropriate volume was added to each proteinformulation to obtain a final concentration of 0.005%.

Formulation Robustness Studies—Stability Study

Based on the initial results obtained from screening of differentbuffers and pH values, three solution compositions were selected forlong term stability studies (sample preparation as in Table 8. A oneyear study was initiated in the proposed formulations (Table 9). Thestability samples at each time point were analyzed by SEC-HPLC, RP-HPLC,OD320, protein concentration, pH, specific activity, SDS-PAGE(Coomassie), and appearance.

TABLE 9 Formulations for Long Term Stability Studies FormulationComposition Formulation with 0.005% Polysorbate 20 Study Conditions A154 mM NaCl, pH 5.9 5° C., 25° C., B  5 mM sodium phosphate, 40° C., and145 mM NaCl, pH 6.0 frozen baseline C 154 mM NaCl, pH 6.5 at ≦−65° C.

TABLE 10 STABILITY OF SELECTED FORMULATIONS AFTER 2 WEEKS AT 40 ± 2° C.Protein SEC-HPLC SEC-HPLC Specific Conc. (% main peak) (% main peak)RP-HPLC Activity Formulation Appearance (mg/mL) OD320 at pH 5.5 at pH7.0 (% main peak) pH (U/mg) Saline, pH 5.9 Baseline Clear to 29.90.044 >99.9 99.7 99.8 5.6 74 slightly opalescent Stressed Clear to 31.10.062 99.8 99.6 99.9 5.7 88 slightly opalescent Saline, pH 7.0 BaselineClear to 29.0 0.038 >99.9 99.6 >99.9 6.7 83 slightly opalescent StressedClear to 32.1 0.041 99.1 99.7 97.0 6.5 66 slightly opalescent 5 mM PBS,pH 6.0 Baseline Clear to 29.8 0.058 >99.9 99.7 99.9 5.9 102 slightlyopalescent Stressed Clear to 30.5 0.076 98.8 99.7 99.7 5.9 95 slightlyopalescent 5 mM PBS, pH 7.0 Baseline Clear to 29.7 0.035 >99.999.7 >99.7 6.9 86 slightly opalescent Stressed Slightly 30.5 0.041 95.499.4 98.0 6.8 94 opalescent to opalescent 1 mM PBS, pH 7.0 with 2 mMCaCl₂, pH 7.0 Baseline Clear to 27.5 0.040 >99.9 99.7 >99.9 5.6 90slightly opalescent Stressed Slightly 27.7 0.042 94.8 99.8 99.0 6.6 93opalescent to opalescent

No significant change in specific activity was observed for the stresssamples (Table 10). Analysis by size exclusion HPLC detected somedegradation for the 2 week thermal stressed sample formulated in 5 mMsodium phosphate with 154 mM NaCl at pH 7.0. The degradation was moreevident by SEC-HPLC using a pH 5.5 mobile phase condition which inducesassociation of rhASA to an octamer. Under these mobile phase conditions,rhASA formulated at pH 7.0 in 1 mM PBS with 2 mM CaCl₂ also exhibitedsignificant degradation.

Following exposure to 1 month at 40° C., samples formulated in 5 mM PBS,pH 7.0 and 1 mM PBS, pH 7.0 with 2 mM CaCl₂ demonstrated fragmentationby SDS-PAGE (data not shown). Consistent with this observation, areduction in the percent main peak was also observed by RP-HPLC andSEC-HPLC for samples stored in these two pH 7 formulations (Table 11). Adecrease in specific activity, however, was only observed for rhASAformulated in 5 mM PBS, pH 7.0.

TABLE 11 Stability of Selected IT Formulations after 1 Month at 40 ± 2°C. Protein SEC-HPLC SEC-HPLC Specific Conc. (% main peak) (% main peak)RP-HPLC Activity Formulation Appearance (mg/mL) OD320 at pH 5.5 at pH7.0 (% main peak) pH (U/mg) Saline, pH 5.9 Baseline Clear to 29.90.044 >99.9 99.7 99.8 5.6 74 slightly opalescent Stressed Clear to 28.30.061 >99.9 99.5 99.9 5.7 107 slightly opalescent Saline, pH 7.0Baseline Clear to 29.0 0.038 >99.9 99.6 >99.9 6.7 83 slightly opalescentStressed Clear to 25.7 0.189 95.7 99.8 99.5 6.6 100 slightly opalescent5 mM PBS, pH 6.0 Baseline Clear to 29.8 0.058 >99.9 99.7 99.9 5.9 102slightly opalescent Stressed Clear to 28.0 0.059 >99.9 99.6 99.9 6.0 94slightly opalescent 5 mM PBS, pH 7.0 Baseline Clear to 29.7 0.035 >99.999.7 >99.9 6.9 86 slightly opalescent Stressed Slightly 27.3 0.142 91.889.6 97.1 6.9 48 opalescent to opalescent 1 mM PBS, pH 7.0 with 2 mMCaCl₂ Baseline Clear to 27.5 0.040 >99.9 99.7 >99.9 5.6 90 slightlyopalescent Stressed Slightly 28.3 0.053 90.6 88.7 97.9 6.7 133opalescent to opalescent

After 3 months storage at 2-8° C., rhASA retained its activity in allformulations (Table 12). Additionally, rhASA maintained >99.8% of itsmain peak area as assessed by SEC-HPLC under both mobile phaseconditions. The stability data for 3 months at 2-8° C. are summarized inTable 12.

TABLE 12 STABILITY OF SELECTED IT BUFFERS AFTER 3 MONTH AT 2-8° C. SEC-SEC- HPLC HPLC (% (% RP- main main HPLC Protein peak) peak) (% SpecificConc. at pH at pH main Activity Formulation Appearance (mg/mL) OD320 5.57.0 peak) pH (U/mg) Saline, pH 5.9 Baseline Clear to 29.9 0.044 >99.999.7 99.8 5.6 74 slightly opalescent Stressed Clear to 29.4 0.05699.8 >99.9 99.9 5.6 97 slightly opalescent Saline, pH 7.0 Baseline Clearto 29.0 0.038 >99.9 99.6 >99.9 6.7 83 slightly opalescent Stressed Clearto 25.5 0.040 99.8 >99.9 >99.9 6.6 127 slightly opalescent 5 mM PBS, pH6.0 Baseline Clear to 29.8 0.058 >99.9 99.7 99.9 5.9 102 slightlyopalescent Stressed Clear to 29.9 0.045 99.8 >99.9 >99.9 5.9 109slightly opalescent 5 mM PBS, pH 7.0 Baseline Clear to 29.7 0.035 >99.999.7 >99.9 6.9 86 slightly opalescent Stressed Clear to 29.0 0.03899.8 >99.9 >99.9 6.9 110 slightly opalescent 1 mM PBS, pH 7.0 with 2 mMCaCl₂ Baseline Clear to 27.5 0.040 >99.9 99.7 ?99.9 5.6 90 slightlyopalescent Stressed Clear to 28.0 0.042 99.8 99.9 >99.9 6.6 105 slightlyopalescent

RhASA formulated in saline, pH 7.0 and 1 mM PBS, pH 7.0 with 2 mM CaCl₂were also evaluated after 3 months storage at the accelerated conditionof 25° C. As shown in FIG. 7, rhASA undergoes a slight amount offragmentation in these formulations (with intensity approximately thatof the 0.5% BSA impurity spike).

Collectively, the preformulation studies demonstrated that the stabilityof rhASA is maintained at pH values in the range of 5.5 to 6.0. In allstudies using formulation solutions at pH 7.0, rhASA demonstratedfragmentation as one of its degradation pathways. The thermal stressresults obtained for the IT formulation candidates at pH 7.0 weresimilar to the thermal stress results obtained for the IV formulations(10 mM sodium citrate-phosphate with 137 mM NaCl) at pH 7.0, wherefragmentation was also observed. Based on these studies, three followingformulations, as in Table 9, were selected for long term stabilitystudies.

Freeze-Thaw Studies

Freeze-thaw experiments were conducted by performing three cycles ofcontrolled freeze-thaw, from ambient to −50° C. at 0.1° C./min on theshelves of a Vertis Genesis 35EL lyophilizer. One mL aliquots of drugsubstance formulated at 30 mg/mL in each of the five solutioncompositions (Table 8) were dispensed into 3 mL glass vials for thisstudy.

Drug substance (38±4 mg/mL) was used for all freeze-thaw studies. Forsmall scale controlled rate freeze-thaw experiments, 2 mL aliquots ofdrug substance were dispensed into 5 mL glass vials with 20 mm Fluorotecstoppers. Freeze-thaw stress experiments were conducted either on theshelves of a Virtis Genesis 35EL lyophilizer or on the shelves of acontrolled rate freezer (Tenney Jr Upright Test Chamber, Model:TUJR-A-VERV). Three cycles of freezing to −50° C. and thawing to 25° C.were performed at either a freeze and thaw rate of 0.1° C./min (using acontrolled rate freezer) or a freeze rate of 0.1° C./min and thaw rateof 0.03° C./min (using lyophilizer). For bulk freeze-thaw studies, 90 mLof drug substance was dispensed into 250 mL polycarbonate bottles. Forfreeze-thaw studies on dry ice, 3 mL of drug substance was dispensedinto 5 mL polycarbonate (Biotainer P/N 3500-05) vials with and withoutpolypropylene screwcaps. The samples were frozen overnight at ≦−65° C.and then placed on dry ice in a closed bucket. For these experiments,stoppered glass vials containing the same sample volume were used as astudy control. For freeze-thaw studies of the diluted drug substance, 1mL aliquots of 1 and 5 mg/mL were dispensed into 2 mL polypropylenetubes and were frozen at ≦−65° C. The frozen samples were subsequentlythawed on the bench top. The cycle was repeated up to 10 times to mimicany potential stress which may occur with handling of the referencestandard aliquots.

The effect of freeze-thaw on the quality of rhASA in the proposedformulations with 0.005% P20 was determined after 3 cycles of controlledrate freezing and thawing (0.1° C./min). No change in the appearance ofrhASA was observed and no soluble aggregates or degradents wereidentified using either SEC or RP-HPLC methods. Additionally, nofragmentation or aggregation bands were observed in the reduced SDS-PAGEanalysis (data not shown). Table 13 summarizes the results of thesestudies.

TABLE 13 Effect of Small Scale Freeze-Thaw on the Quality of rhASA DrugSubstance SEC- SEC- HPLC (% HPLC RP- Protein main (% main HPLC SpecificConc. peak) at peak) at (% main Activity Formulation Appearance (mg/mL)pH 5.5 pH 7.0 peak) pH (U/mg) Saline, pH 5.9 Baseline Clear to 29.9 NT*NT NT 5.6 102 slightly opalescent Stressed Clear to 29.4 >99.9 99.6 99.45.5 86 slightly opalescent Saline, pH 7.0 Baseline Clear to 29.0 NT NTNT 6.7 94 slightly opalescent Stressed Clear to 25.0 >99.9 99.6 99.2 6.696 slightly opalescent 5 mM PBS, pH 6.0 Baseline Clear to 29.8 NT NT NT5.9 92 slightly opalescent Stressed Clear to 31.1 >99.9 99.7 99.5 5.9 95slightly opalescent 5 mM PBS, pH 7.0 Baseline Clear to 29.7 NT NT NT 6.999 slightly opalescent Stressed Clear to 29.9 >99.9 99.6 99.0 6.9 112slightly opalescent 1 mM PBS, pH 7.0 with 2 mM CaCl₂ Baseline Clear to27.5 NT NT NT 5.6 90 slightly opalescent Stressed Clear to 27.3 >99.999.6 99.3 6.7 103 slightly opalescent *Not tested

The results of the small scale controlled rate freeze-thaw studiesperformed in triplicate on 2 mL aliquots of drug substance aresummarized in Table 14. No change in the quality of the drug substancewas observed. The appearance of the frozen and thawed drug substance wascomparable to the appearance of the baseline sample. No reduction inprotein concentration or the purity of material was observed.

TABLE 14 EFFECT OF SMALL SCALE FREEZE-THAW ON THE QUALITY OF RHASA DRUGSUBSTANCE 0.1° C./min Freeze- 0.1° C./min Thaw 0.1° C./min Freeze- UsingControlled 0.03° C./min Thaw Freeze/Thaw Rate Baseline Rate FreezerUsing Lyophilizer Appearance Slightly Slightly opalescent Slightlyopalescent opalescent to to opalescent to opalescent opalescent ProteinConc. (mg/mL) 42 37 36 Optical Density at 320 nm 0.044 0.045 0.043SEC-HPLC (% main peak) 99.6% 99.7% 99.7% RP-HPLC (% mainpeak) >99.9% >99.9% >99.9% pH 5.9 5.9 5.9 Specific Activity (U/mg) 65 6971

All experiments demonstrated that rhASA maintains its quality attributesafter freeze-thaw. It should be noted that a small decreasing trend wasobserved in the activity and the reversed phase percent main peak for 1mg/mL rhASA samples after ten cycles of freeze-thaw as shown in Table15.

TABLE 15 EFFECT OF SMALL SCALE FREEZE-THAW ON RHASA DRUG SUBSTANCEDILUTED TO 1 MG/ML 1 F/T 3 F/T 5 F/T 10 F/T Sample Baseline cycle cyclescycles cycles Protein Conc. (mg/mL)  1.0  1.0  1.0  1.0  1.0 OpticalDensity at 320 nm  0.013  0.005  0.010  0.006  0.017 SEC-HPLC (% mainpeak) 99.5% 99.5% 99.5% 99.5% 99.6% RP-HPLC (% main peak) 99.2% 99.2%99.1% 99.0% 98.9% pH  5.8  5.8  5.8  5.8  5.8 Specific Activity (U/mg)78 76 75 69 65

Agitation Studies

Aliquots of 1.0 mL of sterile filtered protein formulated at 30 mg/mL ineach of five selected solution compositions (Table 8) with P20 weredispensed into 3 mL glass vials with 13 mm Fluorotec stoppers. Vialswere placed on their side on a Labline Orbital Shaker and shaken for 24hours at 100 rpm. The setting was then increased to 200 rpm for the next24 hours of shaking period.

In order to assess the susceptibility of rhASA to agitation, shaking andstirring studies were performed for both drug substance and drug productat concentrations of 35.4 and 30 mg/mL, respectively. For these studies,1.0 mL aliquots of drug substance were dispensed into 3 mL glass vialswith 13 mm Fluorotec stopper. The agitated vials were inspected everyother hour for the first 8 hours and thereafter at 24 and 48 hours. Thevials were removed at the first sign of cloudiness and analyzed. Theappearance of samples was documented and the samples were assayed usingpH, SEC-HPLC, specific activity, and OD320. Drug product agitationstudies were conducted in triplicate (in 154 mM NaCl, pH 6.0 with 0.005%P20) and compared with one replicate of drug substance (in 154 mM NaCl,pH 6.0). Shaking studies were also repeated without inclusion of P20 insaline formulation. For these studies, either 1 mL or 3 mL aliquots ofdrug product at 30 mg/mL were dispensed into 3 mL vials to investigatethe effect of shaking as well as the headspace volume on quality ofrhASA. For these shaking studies, a speed of 220 rpm was used.

Initial shaking studies of rhASA for IV formulation development studiesperformed demonstrated the potential advantage for the presence of asurfactant. For IT formulation development, 0.005% P20 was selected andincluded in formulations for the shaking studies. After 15-24 hours ofshaking at 100 rpm, no visual changes were observed for any of theformulations and the shaking speed was increased to 200 rpm. No changein the appearance of the shaken samples in the proposed candidateformulations was observed after a total of 48 hours of shaking at 100and 200 rpm. The samples were analyzed after this period and the resultsare summarized in Table 16. No changes were observed by any of theassays. SDS-PAGE Coomassie also exhibited no additional high or lowmolecular weight bands for the shaken samples (data not shown).

TABLE 16 RESULTS OF SHAKING STUDIES OF SELECTED IT FORMULATIONS RP-Protein SEC-HPLC (% HPLC Specific Conc. main peak) at (% main ActivityFormulation Appearance (mg/mL) OD320 pH 5.5* peak) (U/mg) Saline, pH 5.9Baseline Clear to 29.9 0.044 NT** NT 111 slightly opalescent StressedClear to 28.5 0.041 >99.9 99.9 111 slightly opalescent Saline, pH 7.0Baseline Clear to 29.0 0.038 NT NT 115 slightly opalescent StressedClear to 24.7 0.032 >99.9 >99.9  110 slightly opalescent 5 mM PBS, pH6.0 Baseline Clear to 29.8 0.058 NT NT 103 slightly opalescent StressedClear to 30.4 0.047 >99.9 99.9 116 slightly opalescent 5 mM PBS, pH 7.0Stressed Clear to 29.7 0.035 NT NT 92 slightly opalescent Baseline Clearto 26.5 0.029 >99.9 99.9 110 slightly opalescent 1 mM PBS, pH 7.0 with 2mM CaCl₂ Baseline Clear to 27.5 0.040 NT NT 147 slightly opalescentStressed Clear to 27.0 0.038 >99.9 99.9 107 slightly opalescent *Due tocolumn problems the SEC profile of dimeric form, at mobile phase pH of7.0, was not obtained. **Not tested

No change in the appearance of drug substance (in 154 mM NaCl at pH 6.0)or drug product (in 154 mM NaCl, pH 6.0, with 0.005% P20) was observedfor the first 4 hours of stirring. After 6 hours of stirring, both drugsubstance and drug product became slightly cloudy (data not shown). Thecloudiness was more pronounced after 48 hours of stirring when no P20was present in the formulation. Additionally, drug substance and drugproduct exposed to shaking became cloudy after 24 hours. FIG. 8demonstrates the agitation observations after 48 hours.

Table 17 and Table 18 summarize the agitation study observations.

TABLE 17 APPEARANCE OF RHASA DRUG SUBSTANCE AND DRUG PRODUCT (WITH P20)AFTER STIRRING Hours Stirred Drug Substance Stirred Drug ProductBaseline Colorless, opalescent, Colorless, opalescent, free of particlesfree of particles 2 No Change No Change 4 No Change No Change 6 1-2flakes, slightly cloudy Fibrous material, slightly cloudy 8 1-2 flakes,slightly cloudy Fibrous material, slightly cloudy 24 1-2 flakes, verycloudy Fibrous material, cloudy 48 1-2 flakes, very cloudy Fibrousmaterial, very cloudy

TABLE 18 APPEARANCE OF RHASA DRUG SUBSTANCE AND DRUG PRODUCT (WITH P20)AFTER SHAKING Hours Shaken Drug Substance Shaken Drug Product BaselineColorless, opalescent, Colorless, opalescent, free of particles free ofparticles 2 No Change No Change 4 No Change No Change 6 No Change NoChange 8 No Change No Change 24 1-2 flakes 1-2 fibers 48 Fibrousmaterial 1-2 fibers

The agitated samples were also analyzed by OD320, pH, specific activity,RP-HPLC, and SEC-HPLC. The results are presented in Table 19 and Table20. Overall, no significant change was observed in the quality of rhASAafter stirring and shaking, with the exception of the appearance.

TABLE 19 EFFECT OF 48 HOURS OF SHAKING ON DRUG SUBSTANCE AND DRUGPRODUCT Shaken Drug Shaken Drug Substance for Product for Freeze/ThawRate Baseline 48 hrs (n = 1) 48 hrs (n = 3) Optical Density at 320 nm0.080 0.053 0.048 SEC-HPLC (% main peak) 99.7% 99.7% 99.7% RP-HPLC (%main peak) >99.9% >99.9% >99.9% pH 6.0 6.0 5.9 Specific Activity (U/mg)96 71 72

Upon stirring drug product after 6 hours, with 0.005% P20, one of thethree replicate became turbid. This sample was removed and the other twosamples were stirred up to 48 hours. Table 20 demonstrates the averageddata for duplicate samples.

TABLE 20 EFFECT OF 48 HOURS OF STIRRING ON DRUG SUBSTANCE AND DRUGPRODUCT Stirred Drug Stirred Drug Substance for Product for Freeze/ThawRate Baseline 6 hrs (n = 1) 48 hrs (n = 2) Optical Density at 320 nm0.080 0.244 0.103 SEC-HPLC (% main peak) 99.7% 99.7% 99.7% RP-HPLC (%main peak) >99.9% >99.9% >99.9% pH 6.0 6.0 6.0 Specific Activity (U/mg)69 73 73

Based on the results and the visual observations, drug substance anddrug product are not readily susceptible to agitation-induceddegradation since it took ˜4 hours of continuous stirring (at settingnumber 5) and 8 hours of continuous vigorous shaking (at 220 rpm) for achange in appearance to occur.

The shaking studies were repeated with drug product in the absence ofP20. For these studies, each vial was filled with either 1 mL or 3 mL ofdrug product in order to investigate the effect of shaking as well asthe headspace volume on the quality of rhASA. For 1 mL fill in 3 mLvials, no change in the appearance of drug product was observed through8 hours of shaking at 220 rpm (n=2, data not shown). Vials with noheadspace (n=1) demonstrated the formation of small flakes, a few fibersand flocculent matter at a faster rate when compared to the vials with alarger headspace. The 48 hour observations are presented in FIG. 9.

The visual results are also summarized in Table 21 and Table 22.

TABLE 21 APPEARANCE OF DRUG PRODUCT IN THE ABSENCE OF POLYSORBATE 20AFTER 48 HOURS OF SHAKING WITH 1 ML FILL IN 3 ML VIAL Shaken Drug ShakenDrug Control_Shaken Product Product Drug Product MLD-200L-001MLD-200L-003 MLD-200L- Hours without P20 without P20 001 with P20Baseline Colorless, slightly opalescent, essentially free of particles 2No Change No Change No Change 4 No Change No Change No Change 6 NoChange No Change No Change 8 No Change No Change No Change 24 FlocculentSignificant flocculent No Change 48 Flocculent Significant flocculent NoChange

TABLE 22 APPEARANCE OF DRUG PRODUCT IN THE ABSENCE OF POLYSORBATE 20AFTER 48 HOURS OF SHAKING WITH 3 ML FILL IN 3 ML VIAL Control_ShakenDrug Product Shaken Drug Product MLD-200L-001 Hours MLD-200L-001 withoutP20 with P20 Baseline Colorless, slightly opalescent, essentially freeof particles 2 No Change No Change 4 Small flakes, few fibers andflocculent No Change 6 Small flakes, few fibers and flocculent No Change8 Small flakes, few fibers and flocculent No Change 24 Small flakes, fewfibers and flocculent No Change 48 Small flakes, few fibers andflocculent No ChangeNo change in the protein concentration was observed. Additionally, nosoluble aggregates were detected using SEC-HPLC for either the 1 mL or 3mL fill volumes (Table 23 and Table 24). Reduced SDS-PAGE (Coomassie)assay did not detect any high or low molecular weight bands (data notshown).

TABLE 23 RESULTS OF 48 HOURS OF SHAKING ON DRUG PRODUCT IN THE ABSENCEOF POLYSORBATE 20 WITH 1 ML FILL IN 3 ML VIAL Shaken Drug Shaken DrugProduct after Product after Control 24 hrs (n = 2) 48 hrs (n = 2) (n= 1) Assay Baseline without P20 without P20 with P20 Concentration 32.332.9 33.8 31.8 (mg/mL) Optical Density 0.164 0.160 0.163 0.169 at 320 nmSEC-HPLC 99.5 99.5 99.5 99.6 (% main peak) pH 6.1 6.1 6.0 6.0 SpecificActivity 64 63 62 72 (U/mg)

TABLE 24 RESULTS OF 48 HOURS OF SHAKING ON DRUG PRODUCT IN THE ABSENCEOF POLYSORBATE 20 WITH 3 ML FILL IN 3 ML VIAL Shaken Drug Shaken DrugProduct after Product after Control 4 hrs (n = 1) 48 hrs (n = 1) (n = 1)Assay Baseline without P20 without P20 with P20 Concentration 31.02 34.432.1 32.6 (mg/mL) Optical Density 0.152 0.163 0.166 0.151 at 320 nmSEC-HPLC 99.6 99.6 99.6 99.6 (% main peak) pH 6.0 6.0 5.9 6.0 SpecificActivity 70 64 65 71 (U/mg)

Buffering Capacity Studies

For determination of the buffering capacity of rhASA, product wastitrated in triplicate, with either dilute acid or dilute base. Aliquotsof 10 mL of drug substance at either 38 or 30 mg/mL (the latter to mimicdrug product) were placed in a 20 mL glass vial to which a micro stirbar was added. Aliquots of 1 μL of 1N hydrochloric acid (HCl) were addedto the protein solution, the contents mixed, and the pH was recorded.The experiment continued with addition of 1 uL HCl spikes, withoutrinsing the pH probe in between the measurements to avoid any dilution,until an approximate pH of 5.5 was achieved. The experiment wasperformed in triplicate and 5 mM phosphate buffer containing 150 mMsodium chloride, pH 6.0, was titrated side-by-side for comparison.Similarly, drug substance at both concentrations was titrated with 1Msodium hydroxide (NaOH) until a final pH of approximately 6.5 wasachieved. In order to investigate the presence of any residual phosphatein rhASA, drug substance was analyzed by inductively coupling plasmamass spectroscopy (ICP-MS). The buffering capacity of diluted rhASA drugsubstance was also investigated to ensure that the pH value of solutiondid not change upon dilution of protein solution. Diluted samplesranging from 30 mg/mL to 1 mg/mL were prepared in 1.5 mL eppendorf tubesand the pH values were measured at the onset of dilution and after oneweek of storage at 2-8° C.

The results of dilute acid and dilute base titration studiesdemonstrated adequate buffering capacity of rhASA solutions. Fortitration studies using HCl, initially the addition of approximately 2μL of 1 M acid did not alter the pH of either drug substance or thebuffer control. Increasing volumes of acid, however, demonstrated adramatic decline on the pH of buffer compared to rhASA drug substance.After addition of 13 μL of 19 M HCl, the pH of the buffer control wasmore than 2 pH units lower than the pH of drug substance. A drugsubstance concentration of 30 mg/mL was also included in this experimentto mimic the drug product concentration. FIG. 10 illustrates thebuffering capacity of rhASA drug substance compared to 5 mM sodiumphosphate buffer, pH 6.3 with 150 mM sodium chloride when titrated withacid.

The titration of rhASA drug substance with sodium hydroxide demonstratedrelatively different results (FIG. 11) with respect to maintaining thepH. The rate of pH change did not differ substantially between drugsubstance and the buffer control.

Based on the observed results, and without wishing to be bound by anytheory, it is likely that rhASA is contributing to the bufferingcapacity of the solution since aspartic acid, glutamic acid, andhistidine side chains have the ability to act as proton acceptors and/ordonors in order to maintain the solution pH. The buffering capacity ofthis protein was also previously observed during preformulation studieswhen the “pH memory” effect was discovered. The retention of pH has beendemonstrated several times both at the laboratory scale and at the largescale operations. Collectively, the results of these two experimentssuggest that the buffering capacity of rhASA in saline is morepredominant in the acidic direction. According to the literature, thebuffering capacity for the lower pH values is a direct indication oflarger numbers of aspartic acid and glutamic acid residues within agiven protein compared to histidine residues. While not wishing to bebound by any theory, this can indeed be the case for arylsulfatase Awhere there are a total of 45 glutamic as well as aspartic acid residuescompared to 18 histidine residues.

The buffering capacity of drug substance may also be attributable toresidual bound phosphate which was shown to be present in drug substanceusing ICP-MS. Table 25 demonstrates the amount of residual phosphatepresent in three different LSDL drug substance lots. This data alsoconfirms the consistency of the ultrafiltration and diafiltration stepsfor the pilot scale process.

TABLE 25 RESIDUAL AMOUNT OF PHOSPHATE IN DRUG SUBSTANCE PRODUCED IN LSDLrhASA Lot No. Phosphate Concentration (ppm) 001 27 002 31 003 31

In order to further understand the buffering capacity of this protein,the effect of dilution on pH was also investigated. Upon dilution ofrhASA drug substance with saline to lower protein concentrations, nochange in the pH values of drug substance was observed. Subsequently,the diluted drug substances were stored at 2-8° C. for one week, afterwhich the pH measurements were recorded. Table 26 summarizes the data.The results demonstrate that dilution and storage at 2-8° C. have noeffect on the pH values of the diluted drug substance. Theseobservations further support the conclusion of the acid and basetitration studies which demonstrated adequate buffering capacity ofrhASA drug substance formulated in saline.

TABLE 26 PH VALUES OF DILUTED RHASA DRUG SUBSTANCE Drug Substance DrugSubstance pH Value after Target Measured Onset One Week ConcentrationConcentration pH of Storage (mg/mL) Using A280 (mg/mL) Value at 2-8° C.37.0 38.8 6.00 6.20 30.0 33.4 6.07 6.10 25.0 28.3 6.04 6.09 20.0 20.16.02 6.12 10.0 9.2 6.04 6.10 5.0 4.5 6.03 6.11 1.0 1.0 6.00 6.07

During investigation of rhASA dilution and pH, it was observed that theappearance of diluted samples demonstrated a concentration dependentdecrease in opalescence, i.e. rhASA samples with higher concentrationswere more opalescent compared to the samples at lower concentrationswhich had an almost clear appearance. FIG. 12 exhibits the observedappearance of diluted rhASA. The 1 mg/mL rhASA solution demonstrated anappearance similar to water while the 30 mg/mL appearance was assessedto be between either Reference Suspensions II and III or III and IV.

Stability Studies

For stability studies, drug substance was formulated at 38±4 mg/mL in154 mM NaCl, pH 6.0 and drug product was formulated at 30±3 mg/mL in 154mM NaCl, pH 6.0 in the presence and absence of 0.005% polysorbate 20.Aliquots of 1 mL of drug substance were dispensed into 5 mLpolycarbonate bottles with polypropylene screw closures and stored at≦−65° C., −15° C. to −25° C., and 2-8° C. Aliquots of 1.0 to 1.1 mL ofdrug product were dispensed into 3 mL glass vials with 13 mm Fluorotecstoppers and stored at 2-8° C., 25±2° C., and 40±2° C. Drug productvials were stored in the upright orientation for initial stabilitystudies and changed to the inverted orientation for the latter studiesusing drug product without P20. At each time point, stability sampleswere tested by SEC-HPLC, RP-HPLC, OD320, protein concentrations, pH,specific activity, SDS-PAGE (Coomassie), and appearance. Peptide map,glycan map, and percent formylglycine were performed annually.Additionally, the latter assays were also performed for the stressed andaccelerated conditions.

Collectively, the results of preformulation, freeze-thaw, and agitationstudies suggest that only three formulations were suitable for furtherdevelopment. Long term stability studies were initiated in these threeformulations in the presence of 0.005% P20. Table 27, Table 28, andTable 29 summarize the stability data for three formulations at selectedtime points.

TABLE 27 LONG TERM STABILITY AT 2-8° C. FOR RHASA IN 154 MM NACL, PH 5.9Test Baseline 3 m 6 m 11 m Appearance Clear to Clear to Clear to Clearto slightly slightly slightly slightly opalescent opalescent opalescentopalescent Protein Conc. 25.6 24.3 26.5 27.3 (mg/mL) SEC-HPLC (%main >99.9 99.8 99.9 99.8 peak) at pH 5.5 SEC-HPLC (% main 99.1 99.099.4 99.7 peak) at pH 7.0 RP-HPLC (% main 99.6 99.7 99.8 >99.9 peak) pH5.9 6.0 6.0 6.0 Specific Activity 95 79 90 87 (U/mg) SDS-Page Conformsto Conforms Conforms Conforms (Coomassie) reference standard with no newbands with intensity greater than the 1% assay control

TABLE 28 LONG TERM STABILITY AT 2-8° C. FOR RHASA IN 154 MM NACL, PH 7.0Test Baseline 3 m 6 m 11 m Appearance Clear to Clear to Clear to Clearto slightly slightly slightly slightly opalescent opalescent opalescentopalescent Protein Conc. 27.3 26.9 28.1 29.2 (mg/mL) SEC-HPLC (% main99.9 97.5 99.8 >99.9 peak) at pH 5.5 SEC-HPLC (% main 99.4 99.0 99.299.8 peak) at pH 7.0 RP-HPLC (% main 99.6 99.7 99.9 >99.9 peak) pH 6.56.6 6.7 6.5 Specific Activity 112 88 98 86 (U/mg) SDS-Page Conforms toConforms Conforms Conforms (Coomassie) reference standard with no newbands with intensity greater than the 1% assay control

TABLE 29 LONG TERM STABILITY AT 2-8° C. FOR RHASA IN 5 MM PHOSPHATEBUFFER WITH 145 MM NACL, PH 6.0 Test Baseline 3 m 6 m 11 m AppearanceClear to Clear to Clear to Clear to slightly slightly slightly slightlyopalescent opalescent opalescent opalescent Protein Conc. 27.9 27.4 27.129.3 (mg/mL) SEC-HPLC (% main 99.9 97.8 99.8 99.9 peak) at pH 5.5SEC-HPLC (% main 98.9 98.9 99.2 99.9 peak) at pH 7.0 RP-HPLC (% main99.7 99.6 99.8 >99.9 peak) pH 5.9 6.0 6.0 5.9 Specific Activity 87 88 9590 (U/mg) SDS-Page Conforms to Conforms Conforms Conforms (Coomassie)reference standard with no new bands with intensity greater than the 1%assay control

Stability studies, performed for up to 11 months at 2-8° C., suggestedthat the quality of rhASA is maintained in the prototype formulations.Representative size exclusion HPLC profiles of rhASA in saline, pH 5.9are shown in FIGS. 13 and 14. Size exclusion HPLC did not detect anysignificant changes in the association state of rhASA after 11 monthsstorage at 2-8° C.

Overall, the quality of drug product in all three candidate formulationswas maintained after 11 months storage at 2-8° C.

Example 4 Toxicology

This example illustrate repeat dose intrathecal (IT) administration ofrhASA from a toxicology and safety pharmacology perspective over asix-month period. The IT test article for this study was rhASA.Thirty-six male and 36 female cynomolgus monkeys were randomly assignedto five treatment groups. The animals in Group 1 were untreated implantdevice control (port & catheter) and were not dosed with the vehicle ortest article; however, these animals were dosed with 0.6 mL of PBS on aschedule matching the test article dosing schedule. The animals inGroups 2-5 received 0.6 mL IT infusion of 0, 3, 10 or 31 mg/mL of rhASA(total dose of 0, 1.8, 6.0, or 18.6 mg) every other week (i.e. a totalof 12 doses). Animals were necropsied at 6 months (24 hours post last ITdose), and the remaining 4 animals/sex/group were necropsied at the endof a 4-week recovery period. Selected tissues were harvested, saved andexamined microscopically.

In general, the test article related changes could be categorized intotwo major types and were present at all dose levels (1.8, 6.0 and 18.6mg/dose). Increase of infiltrates (of white blood cells, usually with aprominent eosinophilic component) in the meninges, the brain parenchyma,the spinal cord parenchyma, trigeminal ganglion, and occasionally thespinal nerve roots/ganglia (or the epineurium surrounding thosestructures). Without wishing to be bound by any theory, this increasewas interpreted to be due to the presence of the test article (aprotein) in the intrathecal space and in the nervous system tissues.Slight, focal increase of microglial cells in the spinal cord and brainin occasional animals (microgliosis was not observed in any high doseanimals). Without wishing to be bound by any theory, both categories ofmorphologic changes were interpreted to be a response to the presence ofthe test article. There was no evidence of neuronal necrosis in anyanimal. None of the test article related changes were related to anybiologically adverse reactions in the brain, spinal cord, spinal nerveroots or ganglia. Specifically, there was no evidence of neuronalnecrosis or a biologically important glial response. There were no testarticle related lesions in the non-nervous system tissues.

Following a one-month recovery period (a dosing free period), the testarticle related changes had either entirely resolved or were limited toremnants of the prior increase in the inflammatory response associatedwith the presence of the test article. There were no adverse morphologiceffects in the recovery animals. As based on a blinded microscopicexamination assigning a semi-quantitative staining score,immunohistochemical staining for Arylsulfatase A (rhASA; the testarticle) was increased in the brain and spinal cord in various celltypes, except neurons, for all test article treated groups at theterminal sacrifice. This increase was also apparent in the Kupffer cellsof the liver. Following the 1-month recovery period, rhASA staining inthe test article treated animals (all dose groups) had returned tocontrol (device and/or vehicle control) levels. In one low dose recoverymale, there were multiple foci of astrocytosis and neuronal loss,indicating multiple areas of prior ischemia, in the cerebral cortex.Although the exact pathogenesis of these lesions in this animal was notapparent, the lack of similar lesions in any other test article treatedanimals, including the high dose animals that received 10× the dose,indicated these lesions were not related to the test article.

The IT test article for this study was rhASA. Thirty-six male and 36female cynomolgus monkeys were randomly assigned to five treatmentgroups. The animals in Group 1 were untreated implant device control(port & catheter) and were not dosed with the vehicle or test article;however, these animals were dosed with 0.6 mL of PBS on a schedulematching the test article dosing schedule. The animals in Groups 2-5received 0.6 mL IT infusion of 0, 3, 10 or 31 mg/mL of rhASA (total doseof 0, 1.8, 6.0, or 18.6 mg) every other week (i.e. a total of 12 doses).Animals were necropsied at 6 months (24 hours post last IT dose), andthe remaining 4 animals/sex/group were necropsied at the end of a 4-weekrecovery period. Selected tissues were harvested, saved and examinedmicroscopically. The table below reflects the study design as itpertained to the pathology aspect of this study.

At the time of sacrifice, the brain was cut in a brain matrix atapproximately 3 mm coronal slice thickness. The first slice and everysecond slice thereafter were fixed in formalin for histopathologicalevaluation and immunohistochemical analysis. The brain was processed asfull coronal sections. These sections included at a minimum thefollowing brain regions.

-   -   Neocortex (including frontal, parietal, temporal and occipital        cortex): brain sections 1 to 8 (and slice 9 when present)    -   Paleocortex (olfactory bulbs and/or piriform lobe): brain        sections 1 to 3    -   Basal ganglia (including caudate and putamen): brain sections 3        and 4    -   Limbic system (including hippocampus and cingulate gyri): brain        sections 4 and 5    -   Thalamus/hypothalamus and midbrain regions including substantia        nigra: brain sections 4 and 5    -   Cerebellum, pons and medulla oblongata: brain sections 6 to 8        (and slice 9 when present).

The brain sections are listed in the data tables as sections 1 to 8/9 (asection 9 was provided by the testing facility for some animals).Sectioning varied slightly between animals. The brain sections (1through 8/9) provided above were the approximate location of the variousanatomic areas. The brain sections are listed in the data tables asindividual sections, with diagnoses pertinent to that section, tofacilitate potential, future additional slide review (if any). Duringdata interpretation, individual brain anatomic sites (as listed above)were compared in order to identify any unique test article effects (i.e.unique to a particular brain region). At TPS, all brain sections fromall animals were embedded in paraffin, sectioned at 5 microns, stainedwith hematoxylin and eosin (H&E) and examined microscopically. Inaddition, brains from the control and high dose animals were stainedwith Fluoro-Jade B (a stain increasing the sensitivity of evaluating thebrain for neuronal degeneration) and a Bielschowsky's silver stain (aprocedure that allows for direct visualization of axons, dendrites andneuronal filaments) and examined.

The spinal cord (cervical, thoracic and lumber) was cut into onecentimeter sections. The first slice and every other slice thereafterwere fixed in formalin for histopathological evaluation andimmunohistochemical analysis. The spinal cord sections (cervical,thoracic (including the catheter tip) and lumbar) from all animals weresectioned at approximately 5 microns, stained with H&E and examined withtransverse and oblique sections taken at each level. Serial spinal cordsections from the control and high dose groups were additionally stainedwith Bielschowsky's silver stain and anti-GFAP (an immunohistochemicalstain that allows for the direct visualization of astrocytes and theirprocesses).

Dorsal spinal nerve roots and ganglion (taken at mid-cervical,mid-thoracic, and mid-lumbar) were embedded in paraffin, with serialsections stained with H&E. In addition, serial sections from the controland high dose groups were stained with Bielschowsky's silver stain.

For the sciatic, tibial and sural nerve sections from all animals: Alongitudinal section of each nerve was embedded in paraffin, sectionedat approximately 5 microns and stained with H&E. A cross section of eachnerve was post-fixed in osmium, embedded in Spurr's resin, sectioned atapproximately 1 to 2 microns and stained with toluidine blue. Osmiumpost-fixation and resin embedding provides for superior preservation ofthe myelin in peripheral nerves and thus a more detailed examination ofthe nerve.

All tissues collected and gross lesions harvested at necropsy from allanimals were also embedded in paraffin, stained with H&E, and examinedmicroscopically. Histopathological processing and evaluations andimmunohistochemical analyses were performed by TPS.

Arylsulfatase A (rhASA) Staining

Positive control slides were supplied by the study sponsor. The slideswere liver sections from mice injected with rhASA. The positive controlslides all showed ample evidence of rhASA in Kupffer cells (sinusoidalmacrophages) in the liver. The positive control slides are stored withthe other slides from this study. All evaluations of the rhASA stainedsections were initially conducted blinded to the treatment group of theanimal. This was accomplished by having the pathologist initially readthe rhASA stained slides with the animal number on the label obscured(by an assistant with knowledge of the actual animal being evaluated),dictating the score (severity grade) during evaluation, and having thesame assistant immediately record the staining score (severity grade)into the data tables. The animal ID was then verified by both the studyneuropathologist and the assistant to guarantee accurate data entry.This procedure was conducted so as to not introduce any bias into thejudging of the overall intensity of staining with theimmunohistochemical stain for the detection of intracellular rhASA. Therelative degree of staining of neurons, meningeal macrophages,perivascular macrophages and glial cells (astrocytes and microglialcells but likely predominantly microglial cells) was graded in all thebrain and spinal cord sections. The average severity scores at eachbrain and spinal cord level for each group was totaled (by group) andrecorded as a total under the tissue heading Brain, General, rhASAStaining and Spinal Cord, General, rhASA Staining.

In general, rhASA staining in neurons of the brain was a measure of theneurons in the cerebral cortex and other nuclear areas in the brain.rhASA staining in meningeal macrophages was evidence of uptake of thetest article by meningeal macrophages and/or endogenous rhASA inmeningeal macrophages. rhASA staining of perivascular macrophages was ameasure of uptake of rhASA by macrophages in the brain/spinal cord (orendogenous rhASA), although it should be noted that the perivascularspace in the brain and spinal cord (the Virchow-Robins space) iscontinuous with the meninges. In general, the grading of rhASA stainingin the glial cells was predominantly a measure of uptake of the testarticle/penetration of the test article into the gray and/or whitematter, especially of the cerebral cortex (the corona radiata is thewhite matter beneath the cerebral cortex). The rhASA staining in thewhite matter appeared to be in astrocytes and microglial cells.

The following grading scheme was used to score the degree of rhASAstaining the various cell types (neurons, glial cells, macrophages).

Grade Explanation (% of the Possible Cells Stained)

-   -   1 Less than 10%    -   2 Greater than 10 to 25%    -   3 Greater than 25 to 50%    -   4 Greater than 50 to 75%    -   5 Greater than 75%

Note this scheme is not strictly quantitative. It was used as anefficient, semi-quantitative method to assess the brain and spinal cordfor the degree of staining with rhASA. It was noted by the StudyNeuropathologist that not all neuronal areas had equal rhASA staining.It was also noted that there was endogenous neuronal staining in somecontrol animals and that cells of the choroid plexus and neurons of thedorsal root ganglia tended to stain strongly for rhASA even in controlanimals. Staining of the choroid plexus and dorsal root ganglia was notgraded but was noted by the study neuropathologist to be prominent, evenin control animals.

Note: All dose groups: Low Dose=1.8 mg/dose; Mid dose=6.0 mg/dose; Highdose=18.6 mg/dose. There were no test article related lesions in thenon-nervous system tissues except for increased rhASA staining in theliver of all dose groups (male and female; see below).

Terminal Sacrifice Animals (6 Months EOW Dosing): rhASA Stained Sections

There was an increase of rhASA staining in the following tissues/celltypes. When considering a test article effect on the degree of rhASAstaining in a particular cell type in a particular dose group, thestaining levels in the concurrent vehicle control and the device control(sacrificed with the recovery sacrifice animals) were considered forcomparison.

Brain, Meninges, Macrophages (all dose groups, males and females)

-   -   Brain, Perivascular, Macrophages (all dose groups, males and        females)    -   Brain, Glial Cells (all dose groups, males and females)    -   Spinal Cord, Meninges, Macrophages (all dose groups, males and        females)    -   Spinal Cord, Perivascular, Macrophages (all dose groups, males        and females)    -   Spinal Cord, Glial Cells (mid and high dose males and females)    -   Liver, Kupffer Cells (all dose groups, males and females)

Because of endogenous staining, rhASA staining levels in the neurons ofthe brain and spinal cord were the most difficult to specificallydefine. The rhASA staining demonstrated consistently increased levels ofrhASA in the meningeal and brain/spinal cord perivascular macrophagesand also within glial cells. There were no detectable differences ofrhASA staining in neurons between the control and test article treatedanimals.

Recovery Sacrifice Animals (6 Months EOW Dosing Followed by 1 Monthwithout Dosing)

In general, test article related changes were either totally resolved orwere notably diminished in those animals allowed a one-month periodwithout dosing prior to necropsy. The following microscopic changes werepresent at an incidence and/or severity that indicated a possiblerelationship to the test article.

Test Article Related Microscopic Changes (Recovery Animals)

-   -   Brain, Meninges, Infiltrates (mid and high dose groups, both        sexes) (FIGS. 16 and 17)    -   Brain, Meninges, Infiltrates, % Eosinophils (mid dose males;        high dose females)    -   Brain, Perivascular, Infiltrates (mid dose males; high dose        females) (FIG. 18)    -   Brain, Perivascular, Infiltrates, % Eosinophils (mid dose males;        high dose females)    -   Brain, Gray Matter, Infiltrates (all dose groups, both sexes)    -   Brain, Gray Matter Infiltrates, % Eosinophils (low dose males)    -   Brain, Gray Matter, Eosinophils, Necrosis (low dose males)    -   Spinal Cord, Meninges, Infiltrates (mid and high dose males; low        and high dose females)    -   Spinal Cord, Meninges, Infiltrates, % Eosinophils (mid dose        males; low dose females)    -   Spinal Cord, Gray Matter, Infiltrates (low dose females)    -   Spinal Cord, Gray Matter, Infiltrates, % Eosinophils (low dose        females)    -   Dorsal Root Ganglion and Roots, Epineurium, Infiltrates (mid        dose females)    -   Spinal Nerve Roots and Ganglia, Infiltrates, Eosinophils (mid        and high dose males; all doses, females)    -   Trigeminal Ganglion, Infiltrates, Eosinophils (mid dose males        and females)

All these changes were interpreted to represent remnants of theincreased inflammatory changes noted in the terminal sacrifice animals.As in the terminal sacrifice animals, there was no evidence the increaseof inflammatory cell infiltrates still present in some recovery animalsrepresented morphologic changes that were causing any adverse effects.There were no test article related lesions in the non-nervous systemtissues.

Recovery Sacrifice Animals (6 Months EOW Dosing Followed by 1 Monthwithout Dosing): rhASA Staining

There was no indication of increased rhASA staining in the recoverymales or females as compared to the device and/or vehicle controls. Inthe brain of the low, mid and high dose recovery males, there wasactually an indication of decreased rhASA staining in some cell types(this varied among the treatment groups) as compared to the deviceand/or vehicle controls. The reason for this, including whether or notthis was an actual effect, was not apparent. One possible explanationwould be that administration of exogenous rhASA may cause some decreasein endogenous rhASA production. A similar finding was not present in thespinal cord of the males. In the recovery males and females, staining inthe liver was similar to that noted in controls.

In general, the test article related changes could be categorized intotwo major types and were present at all dose levels (1.8, 6.0 and 18.6mg/dose).

Increase of infiltrates (of white blood cells, usually with a prominenteosinophilic component) in the meninges, the brain parenchyma, thespinal cord parenchyma, trigeminal ganglion, and occasionally the spinalnerve roots/ganglia (or the epineurium surrounding those structures).This increase was interpreted to be due to the presence of the testarticle (a protein) in the intrathecal space and in the nervous systemtissues.

Slight, focal increase of microglial cells in the spinal cord and brainin occasional animals (microgliosis was not observed in any high doseanimals). Both categories of morphologic changes were interpreted to bea response to the presence of the test article. There was no evidence ofneuronal necrosis in any animal. None of the test article relatedchanges were related to any biologically adverse reactions in the brain,spinal cord, spinal nerve roots or ganglia. Specifically, there was noevidence of neuronal necrosis or a biologically important glialresponse. There were no test article related lesions in the non-nervoussystem tissues. Following a one-month recovery period (a dosing freeperiod), the test article related changes had either entirely resolvedor were limited to remnants of the prior increase in the inflammatoryresponse associated with the presence of the test article. There were noadverse morphologic effects in the recovery animals.

As based on a blinded microscopic examination assigning asemi-quantitative staining score, immunohistochemical staining forArylsulfatase A (rhASA; the test article) was increased in the brain andspinal cord in various cell types, except neurons, for all test articletreated groups. This increase was also apparent in the Kupffer cells ofthe liver. Following the 1-month recovery period, rhASA staining in thetest article treated animals (all dose groups) had returned to control(device and/or vehicle control) levels. In one low dose recovery male,there were multiple foci of astrocytosis and neuronal loss, indicatingmultiple areas of prior ischemia, in the cerebral cortex. Although theexact pathogenesis of these lesions in this animal was not apparent, thelack of similar lesions in any other test article treated animals,including the high dose animals that received 10× the dose, indicatedthese lesions were not related to the test article. Based strictly onthe gross and microscopic findings (on the paraffin embedded,hematoxylin and eosin stained sections) in this study, the no observedadverse effect level (NOAEL) was 18.6 mg.

Example 5 Pharmakinetic Data 6 Month Animal Data

This example provides interpretive analysis for serum and CSFconcentrations of rhASA and anti-rhASA serum antibodies from NorthernBiomedical Research, Inc.

The objective of the example was to evaluate repeat dose intrathecal(IT) administration of rhASA from a toxicology and safety pharmacologyperspective in juvenile (<12 months of age) cynomolgus monkeys. A totalof 12 doses were given in a six month period. Animals were necropsied 24hours or one-month after the last dose. The study design is shown inTable 30.

TABLE 30 Study Design Study Design No. of Nominal No. of Animals, DoseCon- Animals, 1 Month No. of centration Administered 6 Month RecoveryGroup Animals (mg/mL) Dose (mg) Sacrifice Sacrifice 1 4 M, 4 F DC 0 — 4M, 4 F 2 8 M, 8 F 0 0 4 M, 3 F^(a) 4 M, 4 F 3 8 M, 8 F 3 1.8 4 M, 4 F 4M, 4 F 4 8 M, 8 F 10 6.0 4 M, 4 F 4 M, 4 F 5 8 M, 8 F 31 18.6 4 M, 4 F 4M, 4 F DC = Device Control; Animals in Group 1 were not dosed withvehicle or test article. ^(a)Vehicle Control Animal No. 044 wassacrificed early on Day 50 due to a leaking catheter

Assay Methods—Antibody Analysis

Quantitation of anti-rhASA antibodies in the serum and CSF fromcynomolgus monkeys was conducted using a validated method. Briefly, theassay begins by blocking a MSD streptavidin coated plate, followed byincubation with biotin-labeled rhASA. After a washing step, dilutedsamples, calibrators, and QCs are added to the plate and incubated.After an additional wash step, SULFO TAG-labelled drug is added andincubated. A final wash step is performed and MSD read buffer is added.Plates are read immediately. The signal data in relative luminescenceunits (RLU) are analyzed using SOFTMax Pro templates.

Serum and CSF Concentration

Quantitation of rhASA in the serum and CSF from cynomolgus monkeys wasconducted using a validated method. The method is based on Enzyme-LinkedImmunosorbent Assay (ELISA) technology. Briefly, a microtiter plate iscoated with a rabbit polyclonal antibody (SH040) raised againstrecombinant human Arylsulfatase A (ASA). After incubation with ASAreference standards and test samples, bound ASA protein is detected byhorseradish peroxidase (HRP)-conjugated anti-ASA monoclonal antibody(clone 19-16-3). The plate is then incubated with a substrate for HRP,TMB peroxidase. This enzyme-substrate reaction is stopped by theaddition of 2N sulfuric acid (H₂SO₄) and the absorbance of each well ismeasured at the absorbance wavelength 450 nm with a reference wavelength655 nm. The concentrations of ASA in samples are calculated using therhASA calibration curve in the same plate.

Summaries of serum concentrations of rhASA, CSF concentrations of rhASA,Error! Reference source not found.anti-rhASA serum antibodyconcentrations, anti-rhASA CSF antibody concentrations, and incidence ofantibodies by group and sex are presented in Table 33-39 below.

TABLE 33 Summary of Serum Concentration of rhASA in Cynomolgus MonkeysMale Female Mean SD Mean SD Time point ng/mL ng/mL n ng/mL ng/mL n Group1: Vehicle control Prior to Dose 2 0 0 4 0 0 4 Post Dose 2 0 0 4 0 0 4Prior to Dose 4 0 0 4 0 0 4 Post Dose 4 0 0 4 0 0 4 Prior to Dose 6 0 04 0 0 4 Post Dose 6 0 0 4 0 0 4 Prior to Dose 8 0 0 4 0 0 4 Post Dose 80 0 4 0 0 4 Prior to Dose 10 0 0 4 0 0 4 Post Dose 10 0 0 4 0 0 4 Priorto Dose 12 0 0 4 0 0 4 Post Dose 12 0 0 4 0 0 4 Mid Recovery 0 0 4 0 0 4Recovery Necropsy 0 0 4 0 0 4 Group 2: 0 mg Prior to Dose 2 0 0 8 0 0 7Post Dose 2 0 0 8 0 0 7 Prior to Dose 4 0 0 8 0 0 7 Post Dose 4 0 0 8 00 7 Prior to Dose 6 0 0 8 0 0 8 Post Dose 6 0 0 8 0 0 8 Prior to Dose 80 0 8 0 0 8 Post Dose 8 0 0 8 0 0 8 Prior to Dose 10 0 0 8 0 0 7 PostDose 10 0 0 8 0 0 7 Prior to Dose 12 0 0 8 0 0 7 Post Dose 12 0 0 8 0 08 (Prior to 6-month Necropsy) Mid Recovery 0 0 4 0 0 4 Recovery Necropsy0 0 4 0 0 4 Group 3: 1.8 mg Prior to Dose 2 0 0 8 0 0 8 Post Dose 2 49.246.8 8 40.3 27.3 8 Prior to Dose 4 0 0 8 0 0 8 Post Dose 4 0 0 8 0 0 8Prior to Dose 6 0 0 8 0 0 8 Post Dose 6 0 0 8 0 0 8 Prior to Dose 8 0 08 0 0 8 Post Dose 8 0 0 8 0 0 8 Prior to Dose 10 0 0 8 0 0 8 Post Dose10 0 0 8 0 0 8 Prior to Dose 12 0 0 8 0 0 8 Post Dose 12 0 0 8 0 0 8(Prior to 6-month Necropsy) Mid Recovery 0 0 4 0 0 4 Recovery Necropsy 00 4 0 0 4 Group 4: 6.0 mg Prior to Dose 2 0 0 8 0 0 8 Post Dose 2 173.669.5 8 143.2 89.0 8 Prior to Dose 4 0 0 8 0 0 8 Post Dose 4 17 49 8 63.8119.9 8 Prior to Dose 6 0 0 8 0 0 8 Post Dose 6 0 0 8 0 0 8 Prior toDose 8 0 0 8 0 0 8 Post Dose 8 0 0 8 0 0 8 Prior to Dose 10 0 0 8 0 0 8Post Dose 10 0 0 8 0 0 8 Prior to Dose 12 0 0 8 0 0 8 Post Dose 12 0 0 80 0 8 (Prior to 6-month Necropsy) Mid Recovery 0 0 4 0 0 4 RecoveryNecropsy 0 0 4 0 0 4 Group 5: 18.6 mg Prior to Dose 2 0 0 8 0 0 8 PostDose 2 348.0 272.9 8 562.3 204.3 8 Prior to Dose 4 0 0 8 0 0 8 Post Dose4 105.7 274.6 8 172.0 141.3 8 Prior to Dose 6 0 0 8 0 0 8 Post Dose 620.4 38.4 8 88.6 121.4 8 Prior to Dose 8 0 0 8 0 0 8 Post Dose 8 0 0 854.0 89.4 8 Prior to Dose 10 0 0 8 0 0 8 Post Dose 10 0 0 8 6 18 8 Priorto Dose 12 0 0 8 0 0 8 Post Dose 12 0 0 8 0 0 8 (Prior to 6-monthNecropsy) Mid Recovery 0 0 4 0 0 4 Recovery Necropsy 0 0 4 0 0 4

TABLE 34 Summary of CSF Concentrations in Cynomolgus Monkeys Male FemaleMean SD Mean SD Time point ng/mL ng/mL n ng/mL ng/mL n Group 1: VehicleControl Prior to Dose 2 0 0 4 0 0 4 Post Dose 2 0 0 4 0 0 4 Prior toDose 4 0 0 4 0 0 4 Post Dose 4 0 0 4 0 0 4 Prior to Dose 6 0 0 4 0 0 4Post Dose 6 0 0 4 0 0 4 Prior to Dose 8 0 0 4 0 0 4 Post Dose 8 0 0 4 00 4 Prior to Dose 10 0 0 4 0 0 4 Post Dose 10 0 0 3 0 0 4 Prior to Dose12 0 0 3 0 0 4 Post Dose 12 0 0 3 0 0 4 Mid Recovery 0 0 3 0 0 4Recovery Necropsy 0 0 4 0 0 4 Group 2: 0 mg Prior to Dose 2 0 0 6 0 0 7Post Dose 2 0 0 5 0 0 7 Prior to Dose 4 0 0 5 0 0 6 Post Dose 4 0 0 5 00 5 Prior to Dose 6 0 0 5 0 0 5 Post Dose 6 0 0 5 0 0 5 Prior to Dose 80 0 5 0 0 5 Post Dose 8 0 0 5 0 0 5 Prior to Dose 10 0 0 4 0 0 5 PostDose 10 0 0 4 0 0 5 Prior to Dose 12 0 0 4 0 0 5 Post Dose 12 0 0 5 0 05 (Prior to 6-month Necropsy) Mid Recovery 0 0 2 0 0 3 Recovery Necropsy0 0 4 0 0 4 Group 3: 1.8 mg Prior to Dose 2 42491 59255 7 42217 47300 6Post Dose 2 95886 22626 7 125717 61723 6 Prior to Dose 4 17664 24372 650829 41891 6 Post Dose 4 106783 42823 6 138400 49908 6 Prior to Dose 639400 50105 4 45817 38404 6 Post Dose 6 95275 12836 4 104080 37423 5Prior to Dose 8 25799 31589 4 58086 43821 5 Post Dose 8 148750 34664 4119200 66556 5 Prior to Dose 10 25927 31380 4 30380 30328 5 Post Dose 1089975 29494 4 105200 44603 5 Prior to Dose 12 29746 34267 4 82780 659065 Post Dose 12 32030 39155 7 47331 49015 6 (Prior to 6-month Necropsy)Mid Recovery 0 0 3 0 0 2 Recovery Necropsy 0 0 4 0 0 4 Group 4: 6.0 mgPrior to Dose 2 75203 67002 8 146979 233673 6 Post Dose 2 360000 1792768 267667 103369 6 Prior to Dose 4 58064 77210 8 53285 73340 5 Post Dose4 369250 241251 8 305517 152232 6 Prior to Dose 6 77253 91407 8 97987146762 6 Post Dose 6 418600 200098 5 369000 232238 5 Prior to Dose 866342 80374 5 11592 23072 4 Post Dose 8 329400 209841 5 340500 135128 4Prior to Dose 10 119420 148408 5 74031 104609 2 Post Dose 10 412000149278 5 245500 161927 2 Prior to Dose 12 68651 92902 5 74577 105251 2Post Dose 12 141833 173933 7 58986 99016 4 (Prior to 6-month Necropsy)Mid Recovery 0 0 3 0 NA 1 Recovery Necropsy 0 0 4 0 0 4 Group 5: 18.6 mgPrior to Dose 2 289917 291188 7 201339 250774 8 Post Dose 2 734429298352 7 920143 448409 7 Prior to Dose 4 150238 210302 7 169895 185675 6Post Dose 4 984857 570039 7 965167 425924 6 Prior to Dose 6 265479252067 7 288879 226889 6 Post Dose 6 758143 102009 7 1270000 558533 6Prior to Dose 8 190529 240081 7 196021 199396 6 Post Dose 8 1003429538271 7 989800 585072 5 Prior to Dose 10 176297 272500 7 168864 1910876 Post Dose 10 1013000 390673 7 773400 103717 5 Prior to Dose 12 142334196793 5 430542 436534 6 Post Dose 12 291525 350251 7 252142 381200 6(Prior to 6-month Necropsy) Mid Recovery 0 0 3 0 0 2 Recovery Necropsy 00 4 0 0 4

TABLE 35 Summary of Anti-rhASA Antibody Concentration in Serum MaleFemale Mean SD Mean SD Time point ng/mL ng/Ml n ng/mL ng/mL n Group 1:Vehicle control Predose 2 0 0 4 0 0 4 Predose 4 0 0 4 0 0 4 Predose 6 00 4 0 0 4 Predose 8 0 0 4 0 0 4 Predose 10 0 0 4 0 0 4 Predose 12 0 0 40 0 4 Mid Recovery 0 0 4 0 0 4 Recovery 0 0 4 0 0 4 Necropsy Group 2: 0mg Predose 2 0 0 8 0 0 8 Predose 4 0 0 8 0 0 8 Predose 6 0 0 8 0 0 7Predose 8 0 0 8 0 0 7 Predose 10 0 0 8 0 0 7 Predose 12 0 0 8 0 0 7Necropsy 0 0 4 0 0 4 (24 hr after last dose) Mid Recovery 0 0 4 0 0 4Recovery 0 0 4 0 0 4 Necropsy Group 3: 1.8 mg Predose 2 0 0 8 0 0 8Predose 4 18409 21371 8 27648 37504 8 Predose 6 75913 64863 8 8562579871 8 Predose 8 132163 95576 8 151900 97818 8 Predose 10 392338 6066268 290675 186213 8 Predose 12 499438 735028 8 524438 569523 8 Necropsy261625 157865 4 733550 928411 4 (24 hr after last dose) Mid Recovery339250 265888 4 377175 218955 4 Recovery 712500 1107129 4 295525 1747184 Necropsy Group 4: 6.0 mg Predose 2 0 0 8 0 0 8 Predose 4 30419 30561 864000 89510 8 Predose 6 143693 128094 8 191750 150511 8 Predose 8 325750190651 8 305850 224707 8 Predose 10 669125 515458 8 832188 846241 8Predose 12 946125 651530 8 1060775 1088889 8 Necropsy 713500 598812 41047568 1132048 4 (24 hr after last dose) Mid Recovery 1566000 708132 4975500 1149734 4 Recovery 1113250 554510 4 793000 991450 4 NecropsyGroup 5: 18.6 mg Predose 2 0 0 8 0 0 8 Predose 4 56873 39107 8 3999453411 8 Predose 6 311638 237796 8 193263 208952 8 Predose 8 482875270130 8 399363 360425 8 Predose 10 1006750 857916 8 866875 894776 8Predose 12 1419000 1382276 8 1341500 1373771 8 Necropsy 165000 147463 4407300 268570 4 (24 hr after last dose) Mid Recovery 2884250 1363128 42101500 2090420 4 Recovery 2504250 1118042 4 1506000 1524682 4 Necropsy

TABLE 36 Summary of Anti-rhASA Antibody Concentration in CSF Male FemaleMean SD Mean SD Time point ng/mL ng/mL n ng/mL ng/mL n Group 1: Vehiclecontrol Surgery 0 0 4 0 0 4 Predose 2 0 0 4 0 0 4 Predose 4 0 0 4 0 0 4Predose 6 0 0 4 0 0 4 Predose 8 0 0 4 0 0 4 Predose 10 0 0 4 0 0 4Predose 12 0 0 3 0 0 4 Mid Recovery 0 0 3 0 0 4 Recovery 0 0 4 0 0 4Necropsy Group 2: 0 mg Surgery 0 0 7 0 0 6 Predose 2 0 0 6 0 0 7 Predose4 0 0 5 0 0 6 Predose 6 0 0 5 0 0 5 Predose 8 0 0 5 0 0 5 Predose 10 0 04 0 0 5 Predose 12 0 0 4 0 0 5 Necropsy 0 0 3 0 0 2 (24 hr after lastdose) Mid Recovery 0 NA 1 0 0 3 Recovery 0 0 4 0 0 4 Necropsy Group 3:1.8 mg Surgery 0 0 7 0 0 8 Predose 2 0 0 7 0 0 6 Predose 4 0 0 6 41 1016 Predose 6 685 1317 4 632 1413 5 Predose 8 2238 2596 4 2180 4875 5Predose 10 3393 5038 4 5560 12433 5 Predose 12 6436 8266 4 12700 28398 5Necropsy 14848 12401 4 21442 32382 4 (24 hr after last dose) MidRecovery 29307 40617 3 18700 283 2 Recovery 21060 30010 3 13078 7181 4Necropsy Group 4: 6.0 mg Surgery 0 0 7 0 0 8 Predose 2 0 0 7 0 0 6Predose 4 99 172 7 84 187 5 Predose 6 1117 1862 8 1473 2775 6 Predose 83987 5580 5 20824 27320 4 Predose 10 6600 9679 5 2715 1237 2 Predose 125285 7279 5 955 1237 2 Necropsy 16870 16350 4 63000 63000 3 (24 hr afterlast dose) Mid Recovery 66233 42238 3 16800 NA 1 Recovery 53600 14388 328880 29890 4 Necropsy Group 5: 18.6 mg Surgery 0 0 7 0 0 6 Predose 2 00 7 0 0 8 Predose 4 102 192 7 0 0 6 Predose 6 233 351 7 1506 3234 6Predose 8 3378 5931 7 6367 9865 6 Predose 10 16327 24035 7 19567 27542 6Predose 12 11596 16406 5 15143 24351 6 Necropsy 5168 7427 4 12135 103414 (24 hr after last dose) Mid Recovery 54700 26439 3 46315 62770 2Recovery 50725 29217 4 37790 35967 4 Necropsy

TABLE 37 Serum and CSF Concentrations of rhASA, Male and Female Combined(ng/mL) Serum rhASA CSF rhASA (ng/mL) (ng/mL) Group in total Group intotal Mean SD Mean SD Time point ng/mL ng/mL n ng/mL ng/mL n Group 1:Vehicle control Prior to Dose 2 0 0 8 0 0 8 Post Dose 2 0 0 8 0 0 8Prior to Dose 4 0 0 8 0 0 8 Post Dose 4 0 0 8 0 0 8 Prior to Dose 6 0 08 0 0 8 Post Dose 6 0 0 8 0 0 8 Prior to Dose 8 0 0 8 0 0 8 Post Dose 80 0 8 0 0 8 Prior to Dose 10 0 0 8 0 0 8 Post Dose 10 0 0 8 0 0 7 Priorto Dose 12 0 0 8 0 0 7 Post Dose 12 0 0 8 0 0 7 Mid Recovery 0 0 8 0 0 7Recovery Necropsy 0 0 8 0 0 8 Group 2: 0 mg Prior to Dose 2 0 0 16 0 013 Post Dose 2 0 0 16 0 0 12 Prior to Dose 4 0 0 16 0 0 11 Post Dose 4 00 16 0 0 10 Prior to Dose 6 0 0 15 0 0 10 Post Dose 6 0 0 15 0 0 10Prior to Dose 8 0 0 15 0 0 10 Post Dose 8 0 0 15 0 0 10 Prior to Dose 100 0 15 0 0 9 Post Dose 10 0 0 15 0 0 9 Prior to Dose 12 0 0 15 0 0 9Post Dose 12 0 0 15 0 0 10 (Prior to 6-month Necropsy) Mid Recovery 0 08 0 0 5 Recovery Necropsy 0 0 8 0 0 8 Group 3: 1.8 mg Prior to Dose 2 00 16 42365 51844 13 Post Dose 2 44.7 37.3 16 109654 45639 13 Prior toDose 4 0 0 16 34247 36982 12 Post Dose 4 0 0 16 122592 47311 12 Prior toDose 6 0 0 16 43250 40831 10 Post Dose 6 0 0 16 100167 27992 9 Prior toDose 8 0 0 16 43736 40298 9 Post Dose 8 0 0 16 132333 53926 9 Prior toDose 10 0 0 16 28401 28890 9 Post Dose 10 0 0 16 98433 37220 9 Prior toDose 12 0 0 16 59209 58253 9 Post Dose 12 0 0 16 39092 42786 13 (Priorto 6-month Necropsy) Mid Recovery 0 0 8 0 0 5 Recovery Necropsy 0 0 8 00 8 Group 4: 6.0 mg Prior to Dose 2 0 0 16 105964 157408 14 Post Dose 2158.4 78.7 16 320429 153832 14 Prior to Dose 4 0 0 16 56226 72638 13Post Dose 4 40.6 91.7 16 341936 203284 14 Prior to Dose 6 0 0 16 86139113563 14 Post Dose 6 0 0 16 393800 206033 10 Prior to Dose 8 0 0 1642009 65286 9 Post Dose 8 0 0 16 334333 169995 9 Prior to Dose 10 0 0 16106452 130375 7 Post Dose 10 0 0 16 364429 160707 7 Prior to Dose 12 0 016 70344 87227 7 Post Dose 12 0 0 16 111707 151129 11 (Prior to 6-monthNecropsy) Mid Recovery 0 0 8 0 0 4 Recovery Necropsy 0 0 8 0 0 8 Group5: 18.6 mg Prior to Dose 2 0 0 16 242676 264338 15 Post Dose 2 455.1257.8 16 827286 378379 14 Prior to Dose 4 0 0 16 159311 191264 13 PostDose 4 138.8 213.7 16 975769 488021 13 Prior to Dose 6 0 0 16 276279231010 13 Post Dose 6 54.5 93.8 16 994385 453568 13 Prior to Dose 8 0 016 193064 213058 13 Post Dose 8 27.0 67.1 16 997750 531567 12 Prior toDose 10 0 0 16 172866 228817 13 Post Dose 10 3.2 13 16 913167 319975 12Prior to Dose 12 0 0 16 299538 365275 11 Post Dose 12 0 0 16 273348349718 13 (Prior to 6-month Necropsy) Mid Recovery 0 0 8 0 0 5 RecoveryNecropsy 0 0 8 0 0 8

TABLE 38 Serum and CSF Anti-rhASA Antibody, Male and Female Combined(ng/mL) Serum Anti-rhASA CSF Anti-rhASA Antibody Antibody (ng/mL)(ng/mL) Group in total Group in total Mean SD Mean SD Time Point ng/mLng/mL n ng/mL ng/mL n Group 1: Vehicle control Surgery 0 0 8 Predose 2 00 8 0 0 8 Predose 4 0 0 8 0 0 8 Predose 6 0 0 8 0 0 8 Predose 8 0 0 8 00 8 Predose 10 0 0 8 0 0 8 Predose 12 0 0 8 0 0 7 Mid Recovery 0 0 8 0 07 Recovery Necropsy 0 0 8 0 0 8 Group 2: 0 mg Surgery 0 0 13 Predose 2 00 16 0 0 13 Predose 4 0 0 16 0 0 11 Predose 6 0 0 15 0 0 10 Predose 8 00 15 0 0 10 Predose 10 0 0 15 0 0 9 Predose 12 0 0 15 0 0 9 Necropsy 0 08 0 0 5 (24 hr after last dose) Mid Recovery 0 0 8 0 0 4 RecoveryNecropsy 0 0 8 0 0 8 Group 3: 1.8 mg Surgery 0 0 15 Predose 2 0 0 16 0 013 Predose 4 23028 29871 16 21 72 12 Predose 6 80769 70467 16 656 1284 9Predose 8 142031 93979 16 2206 3796 9 Predose 10 341506 436656 16 45979386 9 Predose 12 511938 635340 16 9916 20970 9 Necropsy 497588 666122 818145 22972 8 (24 hr after last dose) Mid Recovery 358213 226397 8 2506429302 5 Recovery Necropsy 504013 766860 8 16499 18552 7 Group 4: 6.0 mgSurgery 0 0 15 Predose 2 0 0 16 0 0 13 Predose 4 47209 66899 16 93 17012 Predose 6 167721 137276 16 1269 2205 14 Predose 8 315800 201572 1611470 19344 9 Predose 10 750656 682110 16 5490 8143 7 Predose 12 1003450868860 16 4048 6328 7 Necropsy 880534 857199 8 36640 45439 7 (24 hrafter last dose) Mid Recovery 1270750 938646 8 53875 42430 4 RecoveryNecropsy 953125 763122 8 39474 26274 7 Group 5: 18.6 mg Surgery 0 0 13Predose 2 0 0 16 0 0 15 Predose 4 48433 46054 16 55 146 13 Predose 6252450 224723 16 821 2204 13 Predose 8 441119 310702 16 4757 7781 13Predose 10 936813 849893 16 17822 24652 13 Predose 12 1380250 1331905 1613531 20189 11 Necropsy 286150 238760 8 8652 9129 8 (24 hr after lastdose) Mid Recovery 2492875 1686472 8 51346 36819 5 Recovery Necropsy2005125 1347857 8 44258 31114 8

TABLE 39 INCIDENCE OF ANTI-RHASA ANTIBODIES AT NECROPSY SerumAntibody-Positive Animals CSF Antibody-Positive Animals (positive/totaltested) (positive/total tested) M F M F 6-month Recovery 6-monthRecovery 6-month Recovery 6-month Recovery Group Necropsy NecropsyNecropsy Necropsy Necropsy Necropsy Necropsy Necropsy 1 (DC) NA 0/4 NA0/4 NA 0/4 NA 0/4 2 (vehicle) 0/4 0/4 0/4 0/4 0/3 0/4 0/2 0/4 3 (1.8 mg4/4 4/4 4/4 4/4 4/4 3/3 3/4 4/4 IT) 4 (6.0 mg 4/4 4/4 4/4 4/4 4/4 3/32/3 4/4 IT) 5 (18.6 mg 4/4 4/4 4/4 4/4 3/4 4/4 4/4 4/4 IT)

The quantitation limit for rhASA in cynomolgus monkey serum is 39.1ng/mL, and all serum samples from Groups 1 and 2 were below quantitationlimit (BQL), see Table 33. Serum levels of rhASA were tested prior toand at 24 hours after Doses 2, 4, 6, 8, 10, and 12 (6-month necropsy),midway through the recovery period, and prior to the recovery necropsy.rhASA levels were undetectable in Group 3 (1.8 mg/dose), Group 4 (6.0mg/dose), and Group 5 (18.6 mg/dose) prior to Doses 2, 4, 6, 8, 10, and12, After Dose 12, midway through the recovery period, and prior to therecovery necropsy. After Dose 2, the levels of rhASA in serum weredose-related. After Dose 4 (Group 3), Dose 6 (Groups 3 and 4), and Doses8 and 10 (Groups 3 and 4 and Group 5 males), rhASA levels wereundetectable. Serum levels of rhASA declined in Group 4 (6.0 mg/dose)after Dose 4 and in Group 5 (18.6 mg/dose) after Doses 4 and 6 for malesand Doses 4, 6, 8, and 10 for females. This apparent decline in serumrhASA levels may be related to the increasing concentration ofanti-rhASA antibodies. There were no apparent sex differences in serumlevels of rhASA, given the sample variability and small group numbers inthis study.

The quantitation limit for rhASA in cynomolgus monkey CSF is 19.5 ng/mL,and all CSF samples from Groups 1 and 2 were BQL, see Table 34. rhASAwas detectable in CSF prior to and after Doses 2, 4, 6, 8, 10, and 12(6-month necropsy) in all dosed groups. The levels were higher postdose(approximately 24 hours postdose) and were dose related. The levels inCSF were much greater than those in serum. There were no apparent sexdifferences in CSF levels of rhASA, given the sample variability andsmall group numbers in this study. rhASA was not detectable midwaythrough the recovery period and prior to the recovery necropsy in alldosed groups. CSF levels at the Dose 12 (necropsy) collections for rhASAtreated groups were lower than levels postdose 8 and 11. Potentialreasons for lower rhASA levels at necropsy include the larger volumetaken (˜2.25 mL total for cell counts, chemistry, rhASA and anti-rhASAantibody) at necropsy vs. those taken at in-life dosing interval (up to0.5 mL pre- or postdose for rhASA concentration). Additionally, someanimals did not have patent catheters at necropsy, and samples weretaken via a CM tap rather than via the catheter. This route consistentlyyielded lower rhASA concentrations as compared with sampling via thecatheter. This is likely due to the limited rostrocaudal direction ofCSF bulk flow that is acknowledged to occur in vertically-orientedanimals like monkeys and man (e.g., it is well known that constituentsof CSF exhibit marked rostrocaudal gradients throughout an individualslifetime).

Anti-rhASA antibodies in serum were detected in every animal treatedwith rhASA at some time point, see Table 35. Animals are defined aspositive for anti-rhASA antibodies if the level of anti-rhASA antibodywas above the quantitation limit (78.1 ng/mL). Animals remained positivefor anti-rhASA antibodies once they seroconverted. No animals werepositive for anti-rhASA antibodies at the predose 2 timepoint. All rhASAanimals except Male No. 026 (Group 4; 6.0 mg/dose) were positive forserum anti-rhASA antibodies at the predose 4 timepoint. Male No. 026 waspositive for serum antibody at the predose 6 timepoint. In Group 5 (18.6mg/kg), the necropsy antibody samples had lower antibody levels. Thisapparent decrease may be due to the presence of rhASA interfering withthe assay. The titer was generally higher in the mid- and high-dosegroups (6.0 and 18.6 mg/dose) than the low dose animals (1.8 mg/dose).The presence of anti-rhASA antibodies is an expected result fromtreating cynomolgus monkeys with a recombinant human protein^(i). Giventhe variability in the results, there was no apparent sex differences.

All animals with detectable anti-rhASA antibodies in CSF had detectablerhASA antibodies in serum as well, with the exception of Female Nos. 049(Group 3; 1.8 mg/dose) and 057 (Group 4; 6.0 mg/dose). The variabilityin the antibody concentration and incidence precludes determination of adose response. Animals are defined as positive for anti-rhASA antibodiesif the level of anti-rhASA antibody was above the quantitation limit(78.1 ng/mL)

Combined values for males and females for serum and CSF rhASA levels andfor anti-rhASA antibodies are shown in Table 36 and Table 37. Combinedmale and female results are similar to the individual sexes, discussedabove.

Example 6 Efficacy

In this example, 11 Wild-type control (mASA +/+hASA −/−) mice wereassigned to Group A and received no treatment. thirty-four (34)hASAC69S/ASA −/− mice were assigned to each of 5 dose groups andreceived vehicle (Group B) or rhASA (rhASA) at doses of 20 mg/kg(intravenous [IV]; Group C) or 0.04, 0.12, and 0.21 mg (Groups D, E, andF, respectively) on Days 1, 9, 15/16, and 22. All IV doses wereadministered via a tail vein. All intrathecal (IT) doses wereadministered as an infusion in a volume of 12 μL at an approximate rangeof 2 μL/20 seconds (Table 40).

TABLE 40 STUDY DESIGN Dose in Total No. mg/kg No. of of brain GroupAnimals Animal Type Treatment Dose Route Injections Sacrifice weight^(a)A 11 Wild-type control None NA NA NA NA NA (mASA +/+ hASA −/−) B 9hASAC69S/ Vehicle Vehicle IT 4 24 hours  0 ASA −/− Control lumbar (Days1, 9, after the C 5 rhASA   20 mg/kg IV (tail 15/16^(b), and fourth NAvein) 22) dose D 5 rhASA 0.04 mg IT 100 lumbar E 5 rhASA 0.12 mg IT 300lumbar F 10 rhASA 0.21 mg IT 520 lumbar NA = not applicable; IT =intrathecal; IV = intravenous. ^(a)Brain weight for mice isapproximately 0.0004 kg. ^(b)Groups C, D, and E were dosed on Day 15;Groups B and E were dosed on Day 16.

The ASA knockout mouse hASAC69S/ASA(−/−) is an accepted model of MLD,and has been used to test potential treatments for this disease. Theintrathecal route is the intended route of administration in humans. Theintravenous route of administration has been tested for this compoundand a similar compound in MLD mice. An intravenous control group hasbeen added as a positive control for histological changes expected inperipheral organs. Animals received 100, 300, or 520 mg/kg of brainweight (0.04, 0.12, 0.21 mg, respectively) of rhASA. The dose levelsnormalized to brain weight selected for this study correspond to dosesthat are planned for use in humans or have been used in toxicologystudies or in previous efficacy models of lysosomal storage diseases.These doses were not expected to have any toxicity.

Receipt

Species Mice (Mus musculus) Strain hASAC69S/ASA (−/−) mice and wild typecontrols Age Approximately 14-17 months at arrival No. of Groups 6 No.of Animals 34 ASA knockout mice + 11 wild type controlsFollowing arrival, each animal was examined to assess health status.

Housing

Animals were group housed in high-temp polycarbonate filter-top cages,with C are Fresh paper bedding and water bottles. Each cage was clearlylabeled with a cage card indicating project, group and animal numbers,and sex. Each animal was uniquely identified using an ear punch system.Animals were treated in compliance with federal guidelines.The targeted conditions for animal room environment and photoperiod wereas follows:

Temperature 22° C. ± 3° C. Humidity 50% ± 20% Light cycle 12 hours lightand 12 hours dark

During and following the dose administration, the photoperiod may havebeen temporarily interrupted for scheduled activities. Suchinterruptions are not considered to affect the outcome or quality ofthis research.

All available wild type animals (11) were assigned to Group A and werenumbered 35 through 45. ASA (−/−) hASA (+/−) animals were assignedconsecutive numbers (1 through 34) as they were removed from theircages, weighed, and ear punched during acclimation. Animals were thenassigned to the treatment groups using Research Randomizer(www.randomizer.org) on Jan. 3, 2011. the first 9 numbers were assignedto Group B, the next 5 to Group C, the next 5 to Group D, the next 5 toGroup E, and the final 10 to Group F. Animals were assigned as followsin Table 41:

TABLE 41 ANIMAL ASSIGNMENT Group N Animal Numbers A 11 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45 B 9 7, 13, 17, 22, 23, 24, 28, 29, 30 C 5 6,16, 19^(a), 21, 32 D 5 5, 9, 14, 18, 27 E 5 1, 2, 4, 8, 11 F 10 3^(b),10, 12, 15, 20, 25, 26, 31, 33, 34 ^(a)Animal No. 19 could not belocated at the time of dosing. ^(b)Animal No. 3 died before dosingbegan.

Test Article and Vehicle

Test Article Identity rhASA Description human recombinant ArylsulfataseA (rhASA) Storage Conditions Approximately 4° C. Vehicle Identity rhASAVehicle (154 mM NaCl, 0.005% polysorbate 20, pH ~6.0) Storage ConditionApproximately 4° C.

Preparation of Vehicle

The vehicle was used as provided. The vehicle was warmed on the benchtop (ambient). Once the vehicle was warmed, the material was mixed bygently swirling and inverting. The bottles were not vortexed or shaken.The bottle was dried before accessing the material. Any remainingvehicle was returned to the refrigerator (1° C.-8° C.).

Dose Formulation Preparation

rhASA was diluted with vehicle to achieve the necessary concentrations.The test article was warmed on the bench top (ambient). Once the testarticle was warmed, the material was mixed by gently swirling andinverting. The bottles were not vortexed or shaken.

Dyes to Track Injections:

An infrared dye (such as IRDye®, LI-COR Biosciences, Lincoln, Nebr.) wasutilized for tracking the injections. Dyes such as this have been usedin intrathecal injections as a survival procedure after intrathecaladministration. The dye was mixed with the test article beforeadministration; 1 nmole of dye in 1 μL was added to the test article. Inaddition to the infrared dye, 1 μL of FD&C blue #1 (0.25%) was used fortracking injections. This blue dye is a common food additive and isgenerally considered safe and non-toxic.

Lumbosacral IT Injection of rhASA or Vehicle

Animals in Groups B, D, E, and F received intrathecal injections on Days1, 9, 15 or 16, and 22.

Adult mice were anesthetized using 1.25% 2,2,2 tribromoethanol (Avertin)at 200-300 μL/10 grams body weight (250-350 mg/kg) by intraperitonealinjection. Dorsal hair was removed between the base of the tail and theshoulder blades using a clippers. The shaved area was cleaned withpovidine/betadine scrub followed by isopropyl alcohol. A small midlineskin incision (1-2 cm) was made over the lumbosacral spine, and theintersection of the dorsal midline and the cranial aspect of the wingsof the ilea (singular ileum) was identified. The muscle in the iliacfossa (gluteus medius) is a heart shaped muscle. The two sides of thetop of the “heart” approximate the location of the wings of the ilea. A32-gauge needle attached to a gas tight 10-20 μL glass Hamilton syringewas inserted until resistance was felt from the underlying bone.Injection of 10 μL of test article, 1 μL of infrared dye, and 1 μL ofFD&C blue #1 (total injection volume of 12 μL) was performed at anapproximate rate of 2 μL/20 seconds (12 μL/2 minutes). The skin incisionwas closed using wound clips. The success of the injection was judged byimaging to determine if the infrared dye had distributed throughout theCNS, as well as the visible blue dye. After imaging, the animal wasallowed to recover in a recovery chamber.

Intravenous Injection of rhASA

Animals in Group C received intravenous injections on Days 1, 9, 15, and22.

For IV injections, animals were anesthetized using isoflurane, ifrequired, and were placed in a restrainer. The tail vein was dilated bywarming by flicking the tail gently with the finger. The injection sitewas then wiped with 70% ethanol. Alternatively, the animal was placed ina warm chamber (40° C.) for 1-1.5 minutes. A 28- to 30-gauge needle wasused to inject test material. The volume of injection was 5-10 mL/kg.

Approximately 24 hours after the fourth dose, animals in Groups B-F wereeuthanized. Animals were subjected to different tissue collectionprocedures, as detailed below. Animals in Group A were not treated;however, they were euthanized on Jan. 27 or 28, 2011 and subjected totissue collection procedures, as detailed below.

Serum (All Animals)

A terminal blood sample (approximately 0.5 mL) was collected from allanimals (Groups A-F) via retroorbital puncture under isofluraneanesthesia. A glass tube was placed in the orbit, gently penetrating thearea behind the eye and thus disrupting the venous drainage locatedbehind the eye. Blood was collected by capillary action and/or gravityflow. Following blood collection, pressure was applied to the orbit tostop the bleeding.

The whole blood samples were processed to serum and frozen at <−80° C.The serum was stored at −80° C. and analyzed for antibodies.

Tissues for Light Microscopy Investigations (Groups A-F; 5 Mice PerGroup)

After blood collection, animals were euthanized via CO₂ asphyxiation. Atail snip was collected prior to perfusion and frozen for possiblegenotyping. The pericardial cavity was exposed. Three (3) mice per groupwere transcardially perfused with heparinized saline solution (1 U/mLsodium heparin in 0.9% NaCl, sterile-filtered) chilled ice-cold and thenwith 4% paraformaldehyde at approximately 4° C. The brain was removed,and the abdomen was cut to expose the internal organs further. The brainand carcass were placed in paraformaldehyde, except for the tail snipwhich was frozen.

Tissues for Lipid Analysis (Groups A, B, and F; 6, 4, and 5 Animals,Respectively)

After blood collection, animals were euthanized via CO₂ asphyxiation. Atail snip was collected prior to perfusion and frozen for possiblegenotyping. The pericardial cavity was exposed. For lipid analyses, 4-6mice per group were transcardially perfused with heparinized salinesolution (1 U/mL sodium heparin in 0.9% NaCl, sterile-filtered) chilledice-cold. Exemplary tissues collected for lipid analyses are presentedin Table 42.

TABLE 42 TISSUES COLLECTED FOR LIPID ANALYSIS Tissues Collected forLipid Analysis Brain (separated into left and right Kidney (2)hemispheres and weighed) Spinal cord (removed from spinal column)Sciatic nerve (2) (dissected free from Tail snip (prior to perfusion)muscle)Upon collection, tissues were weighed and then frozen, either on dry iceor by placing in a −80° C. freezer. The brain was separated into leftand right hemispheres. The right is utilized for lipid analysis by MS.The left will be analyzed for possible N-acetyl-L-aspartate (NAA)analysis. Tissues were stored at −80° C. until analysis (see Table 43).

TABLE 43 SAMPLE STORAGE CONDITIONS Type of Sample Storage TemperatureSerum frozen at circa −80° C. tissues for lipid analysis frozen at circa−80° C. Tail snips frozen at circa −80° C. Tissues for light microscopyApproximately 4° C.

rhASA reduced sulfatide storage in the spinal cord of MLD mice,particularly in the white matter, FIG. 19. Morphometry analysis of thespinal cord demonstrated that the optical density of alcian bluestaining was statistically significantly reduced after rhASA dosing,FIG. 20. rhASA treated MLD mice also exhibited reduced lysosomalactivity in the brain, FIG. 21. This reduction was statisticallysignificant in the high-dose group (0.21 mg-520 mg/kg brain weight)compared with vehicle treated animals, FIG. 22.

Immunotolerant MLD mice (hASAC69S/ASA(−/−)) over 1 year in age receivedintrathecal-lumbar administration of rhASA one time each week for 4weeks (a total of 4 doses). Doses were vehicle (154 mM NaCl, 0.005%polysorbate 20, pH ˜6.0), 0.04, 0.12, 0.21 mg/dose (normalized doseswere 100, 300 and 520 mg/kg of brain weight, respectively). At terminaltimepoints efficacy was evaluated by immunohistochemistry assessment ofsulfatide clearance and lysosome activity within the brain and spinalcord. Spinal cord and brain sections were stained using alcian bluestain targeting sulftatides in tissues Brain sections were also stainedfor the presence of lysosomal-associated membrane protein (LAMP), anindicator of lysosomal processes. Additionally, morphometry analysis wasperformed on alcian blue and LAMP stained sections of the spinal cord(cervical, thoracic and lumbar) and brain.

These preliminary results demonstrate efficacy of intrathecal lumbaradministration of rhASA. Compared to vehicle control mice, rhASA treatedMLD mice exhibit evidence of improvement within the histological markersof disease, such as reduced sulfatide storage (noted by alcian bluestaining) and lysosomal activity in the brain. These histopathologicalchanges were observed near the site of administration (spinal cord) aswell as in the distal portions of the brain.

Example 7 Biodistribution 2 Overview

In this study, 36 male and 36 female juvenile cynomolgus monkeys (<12months at initiation) were assigned to each of 5 dose groups andreceived rhASA (rhASA) at doses of 0 (device control; animals were dosedwith 0.6 mL of PBS), 0 (vehicle control), 1.8, 6.0, or 18.6 mg (Groups1, 2, 3, 4, and 5, respectively) every other week for 6 months for atotal of 12 doses. All doses were administered as an infusion in avolume of 0.6 mL, followed by a flush of 0.5 mL PBS given overapproximately 10 minutes (Table 44Table).

TABLE 44 STUDY DESIGN Study Design No. of Nominal No. of Animals, DoseCon- Animals, 1 Month No. of centration Administered 6 Month RecoveryGroup Animals (mg/mL) Dose (mg) Sacrifice Sacrifice 1 4 M, 4 F DC 0 — 4M, 4 F 2 8 M, 8 F 0 0 4 M, 3 F^(a) 4 M, 4 F 3 8 M, 8 F 3 1.8 4 M, 4 F 4M, 4 F 4 8 M, 8 F 10 6.0 4 M, 4 F 4 M, 4 F 5 8 M, 8 F 31 18.6 4 M, 4 F 4M, 4 F DC = Device Control; Animals in Group 1 were not dosed withvehicle or test article. ^(a)Vehicle Control Animal No. 044 wassacrificed early on Day 50 due to a leaking catheter

Material and Methods Tissue Collection

The brains were cut in a brain matrix at 3 mm thick coronal slicethickness. Each brain was sectioned into full coronal slices including:neocortex (including frontal, parietal, temporal, and occipital cortex),paleocortex (olfactory bulbs and/or piriform lobe), basal ganglia(including caudate and putamen), limbic system (including hippocampusand cingulate gyri), thalamus/hypothalamus, midbrain regions (includingsubstantia nigra), cerebellum, pons, and medulla oblongata. Thelocations from which individual tissue samples were obtained (via 4-mmbiopsy punch) are shown in FIGS. 32-37. The images in FIGS. 32-37 arefrom the University of Wisconsin and Michigan State ComparativeMammalian Brain Collections, (also the National Museum of Health andMedicine). Punch number 22 was not collected, as this structure was notpresent during necropsy. All brain samples were frozen and stored at−60° C. or below prior to analysis for rhASA using an enzyme-linkedimmunosorbent assay.

The first brain slice and every second slice thereafter were fixed informalin for histopathological evaluation and immunohistochemical. Thesecond brain slice and every second slice thereafter were frozen fortest article concentration analysis. Prior to freezing, samples of brainwere taken from the right portion of the even-numbered, test articleanalysis brain slices for biodistribution analysis. The location of thebrain samples were photographed at necropsy and the brain slice numberwas recorded. The samples were obtained using either a 4-mm circularpunch or cut with a scalpel to optimize the amount of white mattercollected. All punches were frozen and stored at −60° C. or below fortest article analysis. The remainder of the brain slice was frozen andstored at −60° C. or below for possible test article analysis. Locationsof the punches are shown in Appendix B.

The spinal cord (cervical, thoracic and lumbar) was cut intoone-centimeter sections. The first slice and every second slicethereafter was fixed in formalin for histopathological andimmunohistochemical analysis. The second slice of spinal cord and everysecond slice thereafter was frozen and stored at −60° C. or lower fortest article analysis. The distribution of slices was adjusted so thatthe slice with the tip of the intrathecal catheter (Slice 0) was fixedin formalin and analyzed for histopathology.

Preparation of Brain, Liver, and Spinal Extracts and Determination ofrhASA Concentration

Brain punches, spinal cord, and liver samples were analyzed using avalidated method in compliance with the United States Food and DrugAdministration (FDA) Good Laboratory Practice (GLP) regulations 21 CFR,Part 58 and with applicable Midwest BioResearch standard operatingprocedures. Tissue samples were homogenized in lysis buffer, centrifugedto remove any tissue debris, and stored at −80° C. until assayed. rhASAconcentration in the soluble fractions of the homogenates was determinedby an ELISA using polyclonal rabbit antibody SH040 as the captureantibody and HRP (horseradish peroxidase)-conjugated anti-ASA monoclonalantibody 19-16-3 as the detection antibody. After a wash step to removeunbound materials, tetramethylbenzidine (TMB) substrate solution reactedwith the peroxide in the presence of HRP-conjugated antibody to producea colorimetric signal that was proportional to the amount of ASA boundby the anti ASA antibody in the initial step. The resulting amount ofrhASA in each tissue homogenate was interpolated from a standard curve.

Samples were also analyzed by a bicinchoninic acid (BCA) proteindetermination assay to obtain the concentration of protein in an unknownsample. The protein concentration for each sample was determined byinterpolation of an albumin standard curve. rhASA concentration resultswere then normalized to total protein in tissue extracts, as determinedby bicinchoninic acid assay.

The ASA levels of all punches for the vehicle, 1.8 mg/dose, 6.0 mg/dose,and 18.6 mg/dose groups are shown in FIG. 23, FIG. 24, FIG. 25, and FIG.26, respectively. The ASA levels of all punches for the recovery animalsfor the device control, vehicle, 1.8 mg/dose, 6.0 mg/dose, and 18.6mg/dose groups are shown in FIG. 27, FIG. 28, FIG. 29, FIG. 30, and FIG.31, respectively

The ASA levels for selected punches that were taken near the surface(meninges) of the brain are shown in FIG. 32. ASA levels for selectedpunches that are considered to contain mostly deep white brain matterare shown in FIG. 33. White matter is composed of bundles of myelinatednerve cell processes (or axons). Selected punches which contain mostlymaterial from the deep grey brain matter are shown in FIG. 34. Greymatter contains neural cell bodies, in contrast to white matter. Thevalues of ASA in selected punches from the surface, deep white and deepgrey are shown for each dose group in FIG. 35.

Spinal cord concentration data is shown in FIG. 36.

Liver concentration data is shown in FIG. 37.

ASA concentration levels in the liver, spinal cord, and brain of thedevice and vehicle-dosed control groups were in some cases measurable.The levels in liver and spinal cord were lower than any of therhASA-treated groups (FIG. 23, FIG. 32, and FIG. 33). The level of rhASAmeasured in the device control and vehicle-dosed animals represents across-reactivity between the anti-rhASA antibody used in the ELISA withthe native cynomolgus monkey protein. The reported values in the devicecontrol and vehicle tissues do not represent quantitative values forcynomolgus monkey rhASA in the tissues, because the degree ofcross-reactivity between the antibody and cynomolgus ASA is not known,and the fact that the assay standards use human ASA. However, withoutwishing to be bound by any theory, the variation in the levels of ASAdetected between device control and vehicle-dosed tissues may beinterpreted as demonstrated variability in the relative amounts ofcynomolgus ASA in different tissues and anatomical regions.

The ASA levels in spinal cord slices ranged from 160-2352, 1081-6607,and 1893-9252 ng/mg protein in males and 0-3151, 669-6637, and1404-16424 ng/mg protein in females for the 1.8, 6.0, and 18.6 mg/dosegroups, respectively (FIG. 32). Levels of ASA were higher in the lumbarregion of the spine than in the cervical region. Levels of ASA proteindetected in the liver were dose responsive in the rhASA treated groupsand were very low in the vehicle group. Mean ASA levels were 88, 674,and 2424 in males and 140, 462, and 1996 ng/mg protein in females forthe 1.8, 6.0, and 18.6 mg/dose groups, respectively (FIG. 33).

Overall, the level of ASA appeared to be dose-related in samplesprepared from the spinal cord slices and liver of the rhASA-dosedgroups. Many of the brain regions tested demonstrated a clear doserelationship between ASA levels and rhASA administration, while otherswere more equivocal. In general, ASA levels in the brain increased withrhASA dose.

Example 8 Pharmacokinetic and Biodistribution Study

The objective this study is to evaluate the pharmacokinetic (PK) andbiodistribution of various therapeutic replacement enzymes afterintrathecal (IT) and intravenous (IV) administration to cynomolgusmonkeys.

In this study, a total of twelve male and twelve female cynomolgusmonkeys with patent intrathecal-lumbar (IT-L) and intrathecal-cisternamagna (IT-CM) catheters were randomly assigned by body weight into fourtreatment groups for Phase 1a (IS2 administration) and Phase 1b (ASAadministration).

Blood and CSF (from IT-CM catheter) were collected at specifiedintervals post dosing for both phases. After the last samples werecollected from Phase 1a, the animals were allowed a 7-day washout periodbefore initiation of Phase 1b.

After the last samples were collected from Phase 1b, the animals will beallowed a 7-day washout period between initiation of Phase 2. A total of12 male and female cynomolgus monkeys from Phase 1b were randomlyassigned by body weight into 12 treatment groups of IS2 (Groups 1a-6a)and ASA (Groups 1b-6b).

The absolute bioavailability of ASA in serum following IT-Ladministration is ˜30 to 40%. In contrast, only 0.5% of the IV dose isbioavailable in CSF.

Exposure to ASA in serum increases in a more than proportional mannerfollowing IT-L administration.

Following IT-L administration, exposure to ASA in CSF increases in aless than proportional manner as dose increases. Summaries of PKparameters of rhASA in serum, PK parameters of rhASA in serum in CSF andbioavailability are shown in Tables 45-47.

TABLE 45 SUMMARY PK PARAMETERS OF ASA IN SERUM OF CYNOMOLGUS MONKEYSSERUM ARYLSULFATASE A ARYLSULFATASE A ARYLSULFATASE A ARYLSULFATASE AARYLSULFATASE A MEAN (PHASE 1B: IV (PHASE 1B: IT-L (PHASE 1B: IT-L(PHASE 1B: IT-L (CV %) 1 MG/KG) 1.8 MG) 6 MG) 18.6 MG) N 8 6 8 8 AUC0-T10505 (16.9) 2219 (41.9) 10352 (31.9) 17583 (28.2) (NG · H/ML) AUC0-∞11069 (17.2)   NC (NC)B  9634 (28.9)C 20789 (27.8)D (NG · H/ML) CMAX11911 (20.0)  363 (40.4)  1160 (29.9)  1621 (25.1) (NG/ML) TMAXA   0.08(0.08, 0.08)  4.00 (2.00, 4.00)   4.00 (1.00, 4.00)   3.00 (1.00, 4.00)(H) T½ (H)   6.55 (31.8)   NC (NC)B   6.77 (21.4)C   7.40 (32.8)D CL OR 261 (17.0)   NC (NC)B  654 (25.0)C  944 (25.4)D CL/F (ML/H) VZ OR  2418(32.4)   NC (NC)B  6523 (41.3)C  9686 (25.8)D VZ/F (ML)

TABLE 46 SUMMARY PK PARAMETERS OF ASA IN CSF OF CYNOMOLGUS MONKEYS CSFArylsulfatase A Arylsulfatase A Arylsulfatase A Arylsulfatase AArylsulfatase A (Phase 1b: IV (Phase 1b: IT-L (Phase 1b: IT-L (Phase 1b:IT-L Mean (CV %) 1 mg/kg) 1.8 mg) 6 mg) 18.6 mg) N 4 6 8 8 AUC0-t (ng ·h/mL)  1629 (179.8) 1267266 (86.6) 5334329 (68.8) 8028775 (71.2) AUC0-∞(ng · h/mL)  8221 (NC)b 1595942 (79.1)c 4291829 (84.2)d 9406664 (64.5)eCmax (ng/mL)   69.3 (94.2)  345167 (48.7) 1039079 (73.6) 1841125 (62.8)Tmaxa (h)   6.00 (1.00, 8.00)    0.08 (0.08, 4.00)    0.29 (0.08, 4.00)   2.04 (0.08, 4.00) t½ (h)   37.6 (NC)b    23.6 (68.3)c    17.1 (31.3)d   13.4 (29.3)e CL or CL/F (mL/h)  392 (NC)b    1.95 (74.1)c    38.1(214.8)d *    3.04 (66.1)e Vz or Vz/F (mL) 21237 (NC)b    80.6 (110.4)c  1090 (215.1)d    67.6 (81.2)e

TABLE 47 BIOAVAILABILITY OF ASA IN SERUM AND CSF AbsoluteBioavailability Comparison Arylsulfatase A Arylsulfatase A (Phase 1b:IT-L Arylsulfatase A (Phase 1b: IT-L 1.8 mg) (Phase 1b: IT-L 6 mg) 18.6mg) Fabs (%) NC 39.9 27.3

The bioavailability of ASA in serum following IT-L administration is˜30-40%. In contrast, only 0.5% of the dose administered by IV route isbioavailable in CSF. CSF serum partition is shown in Table 48.

TABLE 48 CSF: SERUM PARTITION CSF: PLASMA PARTITION ARYLSULFATASE AARYLSULFATASE A ARYLSULFATASE A ARYLSULFATASE A (PHASE 1B: IV (PHASE 1B:IT-L (PHASE 1B: IT-L (PHASE 1B: IT-L 1 MG/KG) 1.8 MG) 6 MG) 18.6 MG)0.74 NC 445 452

Example 9 Treatment of MLD Patients

Direct CNS administration through, e.g., IT delivery can be used toeffectively treat MLD patients. This example illustrates a multicenterdose escalation study designed to evaluate the safety of up to 3 doselevels every other week (EOW) for a total of 40 weeks of rhASAadministered via an intrathecal drug delivery device (IDDD) to patientswith late infantile MLD. Various exemplary intrathecal drug deliverydevices suitable for human treatment are depicted in FIGS. 45-48.

Up to 20 patients will be enrolled:

-   -   Cohort 1: 5 patients (Lowest Dose)    -   Cohort 2: 5 patients (Intermediate Dose)    -   Cohort 3: 5 patients (Highest Dose)    -   5 patients will be randomized to no treatment.

Patients are selected for the study based on inclusion of the followingcriteria: (1) appearance of first symptoms prior to 30 months of age;(2) ambulatory at the time of screening (defined as the ability to standup alone and walk forward 10 steps with one hand held); (3) presence ofneurological signs at time of screening. Typically, patients history ofhematopoietic stem cell transplantation are excluded.

Safety of ascending doses of rhASA administered by IT injection for 40weeks in children with late infantile MLD is determined. In addition,the clinical activity of rhASA on gross motor function, and single andrepeated-dose pharmacokinetics in serum and concentrations incerebrospinal fluid (CSF) are assessed.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds of theinvention and are not intended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications, websites and other reference materials referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference.

1. A stable formulation for intrathecal administration comprising anarylsulfatase A (ASA) protein, salt, and a polysorbate surfactant. 2.The stable formulation of claim 1, wherein the ASA protein is present ata concentration ranging from approximately 0-100 mg/ml.
 3. (canceled) 4.The stable formulation of claim 1, wherein the ASA protein comprises anamino acid sequence of SEQ ID NO:1.
 5. The stable formulation of claim1, wherein the ASA protein is produced from a human cell line.
 6. Thestable formulation of claim 1, wherein the ASA protein is produced fromCHO cells.
 7. The stable formulation of claim 1, wherein the salt isNaCl. 8-10. (canceled)
 11. The stable formulation of claim 1, whereinthe polysorbate surfactant is selected from the group consisting ofpolysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 andcombination thereof.
 12. The stable formulation of claim 11, wherein thepolysorbate surfactant is polysorbate
 20. 13. The stable formulation ofclaim 12, wherein the polysorbate 20 is present at a concentrationranging from approximately 0-0.2%.
 14. (canceled)
 15. The stableformulation of claim 1, wherein the stable formulation further comprisesa buffering agent.
 16. The stable formulation of claim 15, wherein thebuffering agent is selected from the group consisting of phosphate,acetate, histidine, succinate, citrate, Tris, and combinations thereof.17. The stable formulation of claim 1, wherein the buffering agent isphosphate.
 18. The stable formulation of claim 17, wherein the phosphateis present at a concentration no greater than 50 mM.
 19. (canceled) 20.The stable formulation of claim 1, wherein the formulation has a pH ofapproximately 3-8.0. 21-22. (canceled)
 23. The stable formulation ofclaim 1, wherein the formulation is a liquid formulation.
 24. The stableformulation of claim 1, wherein the formulation is formulated aslyophilized dry powder.
 25. The stable formulation of claim 17, whereinthe formulation further comprises a stabilizing agent.
 26. The stableformulation of claim 25, wherein the stabilizing agent is selected fromthe group consisting of sucrose, glucose, mannitol, sorbitol, PEG 4000,histidine, arginine, lysine, phospholipids and combination thereof. 27.A stable formulation for intrathecal administration comprising anarylsulfatase A (ASA) protein, salt, and a buffering agent. 28-36.(canceled)
 37. A container comprising a single dosage form of a stableformulation according to claim
 1. 38. The container of claim 37, whereinthe container is selected from an ampule, a vial, a cartridge, areservoir, a lyo-ject, or a pre-filled syringe. 39-40. (canceled) 41.The container of any one of claims 37-40, wherein the stable formulationis present in a volume of less than about 50.0 mL.
 42. (canceled)
 43. Amethod of treating metachromatic leukodystrophy (MLD) disease comprisinga step of administering intrathecally to a subject in need of treatmenta formulation according to claim
 1. 44. The method of claim 43, whereinthe intrathecal administration of the formulation results in nosubstantial adverse effects in the subject.
 45. The method of claim 44,wherein the intrathecal administration of the formulation results in nosubstantial adaptive T cell-mediated immune response in the subject. 46.The method of claim 43, wherein the intrathecal administration of theformulation results in delivery of the ASA protein to oligodendrocytesof deep white brain matter.
 47. The method of claim 43, wherein the ASAprotein is delivered to neurons, glial cells, perivascular cells and/ormeningeal cells.
 48. The method of claim 43, wherein the ASA protein isfurther delivered to the neurons in the spinal cord.
 49. The method ofclaim 43, wherein the intrathecal administration of the formulationfurther results in systemic delivery of the ASA protein in peripheraltarget tissues.
 50. The method of claim 49, wherein the peripheraltarget tissues are selected from liver, kidney, and/or heart.
 51. Themethod of claim 43, wherein the intrathecal administration of theformulation results in lysosomal localization in brain target tissues,spinal cord neurons and/or peripheral target tissues.
 52. The method ofany claim 43, wherein the intrathecal administration of the formulationresults in reduction of sulfatide storage in the brain target tissues,spinal cord neurons and/or peripheral target tissues.
 53. (canceled) 54.The method of claim 43, wherein the intrathecal administration of theformulation results in reduced progressive demyelination and axonal losswithin the CNS and PNS.
 55. The method of claim 43, wherein theintrathecal administration of the formulation results in increased ASAenzymatic activity in the brain target tissues, spinal cord neuronsand/or peripheral target tissues. 56-59. (canceled)
 60. The method ofclaim 43, wherein the intrathecal administration of the formulationresults in reduced intensity, severity, or frequency, or delayed onsetof at least one symptom or feature of the MLD disease.
 61. The method ofclaim 60, wherein at least one symptom or feature of the MLD disease isincreased intracranial pressure, hydrocephalus ex vacuo, accumulatedsulfated glycolipids in the myelin sheaths in the central and peripheralnervous system and in visceral organs, progressive demyelination andaxonal loss within the CNS and PNS, and/or motor and cognitivedysfunction.
 62. The method of claim 43, wherein the intrathecaladministration takes place at an interval selected from once every twoweeks, once every month, once every two months. 63-64. (canceled) 65.The method of claim 43, wherein the intrathecal administration is usedin conjunction with intravenous administration. 66-67. (canceled) 68.The method of claim 43, wherein the intrathecal administration is usedin absence of intravenous administration.
 69. The method of claim 43,wherein the intrathecal administration is used in absence of concurrentimmunosuppressive therapy.